Accepted Manuscript Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus Carmela Pennacchia , Pieter Breeuwer , Rolf Meyer PII:
S0740-0020(14)00095-1
DOI:
10.1016/j.fm.2014.05.002
Reference:
YFMIC 2161
To appear in:
Food Microbiology
Received Date: 20 August 2013 Revised Date:
29 April 2014
Accepted Date: 2 May 2014
Please cite this article as: Pennacchia, C., Breeuwer, P., Meyer, R., Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus, Food Microbiology (2014), doi: 10.1016/j.fm.2014.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development of a Multiplex-PCR assay for the rapid identification of Geobacillus
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stearothermophilus and Anoxybacillus flavithermus
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Carmela Pennacchia*,a, Pieter Breeuwerb and Rolf Meyera
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a
Nestlé Product Technology Center (PTC), NESTEC Ltd, Nestlé Strasse 3, 3510 Konolfingen, Switzerland.
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b
Nestlé Nutrition, NESTEC Ltd, Avenue Reller 22, 1800 Vevey, Switzerland.
7 *Corresponding author: Tel.: +41 (0)31 790 1815; fax: +41 (0) 31 790 1552
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E-mail address: [email protected]
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ABSTRACT
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The presence of thermophilic bacilli in dairy products is indicator of poor hygiene. Their
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rapid detection and identification is fundamental to improve the industrial reactivity in the
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implementation of corrective and preventive actions.
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In this study a rapid and reliable identification of Geobacillus stearothermophilus and
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Anoxybacillus flavithermus was achieved by species-specific PCR assays. Two primer sets,
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targeting the ITS 16S-23S rRNA region and the rpoB gene sequence of the target species
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respectively, were employed. Species-specificity of both primer sets was evaluated by using
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53 reference strains of DSMZ collection; among them, 13 species of the genus Geobacillus
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and 15 of the genus Anoxybacillus were represented. Moreover, 99 wild strains and 23 bulk
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cells collected from 24 infant formula powders gathered from several countries worldwide
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were included in the analyses. Both primer sets were highly specific and the expected PCR
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fragments were obtained only when DNA from G. stearothermophilus or A. flavithermus was
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used. After testing their specificity, they were combined in a Multiplex-PCR assay for the
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simultaneous identification of the two target species. The specificity of the Multiplex-PCR
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was evaluated by using both wild strains and bulk cells. Every analysis confirmed the reliable
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identification results provided by the single species-specific PCR methodology.
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The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the
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PCR procedures developed in this study highlight the advantage of their application for the
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specific detection and identification of the thermophilic species G. stearothermophilus and A.
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flavithermus.
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Keywords: Geobacillus stearothermophilus, Anoxybacillus flavithermus, thermophilic
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bacilli, milk powder, PCR.
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1. INTRODUCTION
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Thermal processes based on high temperatures are commonly used in food industries to
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guarantee that food products remain stable for long period at ambient temperatures (Prevost et
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al., 2010). However, the thermal treatments are not always sufficient to inactivate all spore-
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forming bacteria, especially those that are highly heat-resistant, but non-pathogenic (Hornstra
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et al., 2009). Among them, the thermophilic bacilli were reported to be important
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contaminants in milk powders (Ronimus et al., 2003; Ruckert at al., 2004, Scott et al., 2007),
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canned foods and dairy products (Denny, 1981; Jay et al., 2005; Scott et al., 2007; Prevost et
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al., 2010).
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In the dairy industry, the thermophilic bacilli can be divided in two main groups: facultative
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thermophiles (also known as thermotolerant microorganisms) and obligate thermophiles. The
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obligate thermophiles grow at elevated temperatures (approximately 48-60°C) and mainly
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include the two species: Geobacillus (G.) stearothermophilus and Anoxybacillus (A.)
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flavithermus (Flint et al., 2001; Ronimus et al., 2003; Scott et al., 2007; Burgess et al., 2010).
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Both species exhibit a fast growth rate and tend to form biofilm on the stainless steel surfaces
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of processing equipment (Scott et al., 2007).
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The presence of the thermophilic bacilli in dairy products is indicator of poor hygiene; high
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counts are unacceptable, since they can lead to product defects caused by the production of
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heat-stable enzymes, such as proteinases and lipases, and acids capable to spoil the final
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product (Chopra and Mathur, 1984; Cosentino et al., 1997; Chen et al., 2004; Gundogan and
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Arik, 2004).
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Identification of the bacteria capable to contaminate milk powders and possibly cause their
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spoilage can help in implementing corrective and preventive actions, in particular at level of
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the manufacturing process steps before heat treatment. Molecular methods able to rapidly
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detect and identify thermophilic contaminants are fundamental to improve the industrial
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reactivity (Prevost et al., 2010).
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In the last few years several efforts were directed toward the development of PCR-based
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methods for the investigation of the thermophilic bacilli contamination in milk powders, but
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most of the studies were focused on their total enumeration by quantitative real-time PCR,
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without distinguishing among the different species. Moreover, some identification methods
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were too expensive or required a lot of expertise to be easily implemented in the routine
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analysis scheme of the industrial laboratories (Flint et al., 2001; Ronimus et al., 2003;
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Rueckert et al., 2005 a-b; Rueckert et al., 2006; Prevost et al., 2010; Postollec et al., 2012).
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The main objective of this study was to develop rapid and easy PCR-based assays for the
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detection and identification of the thermophilic species G. stearothermophilus and A.
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flavithermus. Both species detection was based on activation, germination and outgrowth of
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the spores on agar plates followed by PCR. This step was preferred to a direct PCR detection
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from milk powder samples to prevent false-negative reactions due to inhibitory substances
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present in the milks like calcium ions (Bickley et al., 1996) or proteinases (Powell et al.,
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1994) as well as false-positive reactions due to the free DNA of dead bacteria.
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The G. stearothermophilus species-specific PCR assay described by Prevost et al. (2010) was
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deeply modified. A new primer set for the species-specific identification of A. flavithermus
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was designed by the alignment and comparison of the rpoB gene sequences of Anoxybacillus
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species. Specificity of both PCR assays was validated by testing reference strains and by
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analysing the 16S rRNA sequence of a representative number of wild strains isolated from
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naturally contaminated milk powder samples collected worldwide. The species-specific PCR
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assays were also tested to detect and identify the two target species in the bulk cells collected
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from the same milk powder samples.
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After fulfilling the validation process, the two PCR assays were combined in a unique reliable
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Multiplex-PCR assay, capable to further halve the analysis time and cost.
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2. MATERIALS AND METHODS
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2.1. Bacterial reference strains and growth conditions
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This study involved 53 reference strains from DSMZ collection (Table 1), representing 15
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different species of the genus Anoxybacillus, eight different species of the genus Bacillus,
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four different species of the genus Clostridium, 13 different species of the genus Geobacillus,
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two different species of the genus Moorella, three different species of the genus Paenibacillus
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and two different species of the genus Thermoanaerobacterium.
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With the exception of G. stearothermophilus strains 297, 456, 1550 and 2027, all the
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reference strains were purchased as ready-to-use DNA from DSMZ.
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Working cultures of G. stearothermophilus strains were purchased as lyophilized cultures and
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were grown on Nutrient Agar (NA; Oxoid Ltd., Basingstoke, Hampshire, UK) at 60°C in
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aerobic conditions for 48 h.
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2.2. Isolation of wild strains from infant formula (IF) milk powders
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24 IF milk powder samples (Table 2) were collected worldwide from different factories and
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used to isolate wild strains, naturally contaminating IF powders and resistant to high
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temperature.
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Ten grams of each sample were transferred into a sterile Stomacher bag, reconstituted with 90
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ml of Tryptone Sodium Chloride broth + antifoam (TS+ broth; Tryptone, Oxoid; Sodium
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Chloride, Merck; Silicon antifoam, Sigma) with the addition of 2 g/l of soluble starch (Merck)
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and treated with a Stomacher machine for 30 sec. To induce spore outgrowth, milk
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suspensions were heat-treated at 100°C for 30 minutes in an oil bath and then incubated at
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60°C for 48 h to promote the growth of thermophilic bacilli. After the incubation, serial
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decimal dilutions were prepared by using Maximum Recovery Diluent (MRD; Oxoid) and 0.1
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ml were spread-plated on NA plates in duplicate. Plates were incubated at 60°C for 48 h
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under aerobic conditions.
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After the incubation, the first plate from each sample showing a number of colonies from 20
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to 200 CFU was used to purify and isolate 2-5 colonies showing different morphology, while
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the duplicate plate was used for the collection of bulk cells. The list of the wild strains
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isolated from each milk powder sample is reported in Table 2.
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2.3. Collection of bulk cells
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The cultivable high temperature-resistant microbiota of 23 IF milk powder samples were
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collected from the duplicate NA plates showing 20-200 CFU (Table 2). All colonies present
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on the plates were collected by gently scraping of the surface with a sterile spatula, and each
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was resuspended in 2 ml of MRD and collected in a vial (“bulk cells”). After centrifugation at
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12.000 rpm for 10 min, each pellet was used for DNA extraction.
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For each IF sample, two replicates were prepared; the bulk cells collection from each replicate
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was conducted in duplicate.
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2.4. DNA extraction
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DNA extraction was carried out from the 4 G. stearothermophilus DSM strains 297, 456,
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1550 and 2027, from the pellet of bulk cells and from purified wild strains by using the
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InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA), following the procedure described
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by the supplier. DNA extraction from each bulk cell and from each wild strain was performed
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in triplicate. After extraction, DNA was quantified by using the NanoDrop Lite
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Spectrophotometer (ThermoScientific, Wilmington, DE, USA), standardized at 25 ng/µl by
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using ultra-pure sterile deionized water and 50 ng of DNA were used for PCR amplification.
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2.5. PCR conditions
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To verify that the extracted DNA could be amplified, each wild strain’s DNA was submitted
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to the 16S rRNA amplification by using the universal primers for eubacteria fD1 (5’-AGA 6
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GTT TGA TCH TGG CTC AG-3’) and rD1 (5’-GGM TAC CTT GTT ACG AYT TC-3’)
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(Escherichia coli positions 8–17 and 1540–1524, respectively) described by Weisburg et al.
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(1991). Each DNA was tested for PCR amplification in duplicate. Each PCR mixture (final
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volume, 50 µl) contained 50 ng of template DNA, each primer at a concentration of 0.2
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µΜ, each deoxynucleoside triphosphate at a concentration of 0.25 mM, 2.5 mM MgCl2, 5 µl
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of 10X PCR buffer (Invitrogen, Life Technologies Europe B.V.) and 2.5 U of Taq
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polymerase (Invitrogen). DNA amplification was performed by using the thermocycler
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GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies Europe B.V.). PCR
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conditions consisted of 1 cycle at 94°C for 3 min to denaturate DNA followed by 30 cycles (1
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min at 94°C, 45 sec at 54°C, 2 min at 72°C) plus one additional cycle at 72°C for 7 min as a
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final chain elongation. The presence of PCR products was verified by agarose (1% w/v) gel
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electrophoresis at 7 V/cm for 2 h.
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Species-specific PCR for G. stearothermophilus identification was conducted by using the
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primers Fits2 (5’-GGG GAA GCG CCG CGT TCG G-3’) and Rits2 (5’-GTG CAA GCA
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CCC TTG CAG GCG AAG A-3’), already described by Prevost et al. (2010) and targeting
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the ITS 16S-23S rRNA region. Each PCR mixture (final volume, 50 µl) was prepared as
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described above and PCR conditions were deeply modified, since those described by Prevost
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et al. (2010) were not highly specific for the G. stearothermophilus species only. In particular,
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PCR conditions were shortened by applying 1 cycle at 94°C for 3 min, to denaturate DNA,
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followed by 25 cycles (15 sec at 94°C, 30 sec at 69°C), without elongation step. Each DNA
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was tested for PCR amplification in duplicate. The presence of the 302 bp PCR products was
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verified by agarose (2% w/v) gel electrophoresis at 7 V/cm for 2 h.
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Species-specific PCR for A. flavithermus identification was conducted by using the newly
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designed primers Anx-RpoB5f (5’- TCC GAT TGC GGA AGA TGG GAC G -3’) and Anx-
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RpoB1bisr (5’-GAT ACA CGC TGT GGC TAC CGA T-3’). The primers were designed on
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the basis of the comparison of the rpoB gene sequences of A. flavithermus and of the
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Anoxybacillus species listed in Table 1. Sequence alignment was performed by ClustalW
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program (Thompson et al., 1994). The alignment is showed in Figure 1, where the sequence
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variability used for the species-specific design of the primers is highlighted. The primers were
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targeting the rpoB gene in positions 167-188 and 308-286, respectively and the expected size
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of the PCR product was of 142 bp. PCR mixture and PCR conditions applied were exactly the
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same applied for species-specific identification of G. stearothermophilus.
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2.6. Determination of the species-specific PCR sensitivities
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The sensitivity was determined for each of the two species-specific PCR primer pairs with
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DNA extracted from reference strains A. flavithermus DSM 2641 and G. stearothermophilus
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DSM22.
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The level and purity of DNA of the extracted DNA were evaluated by measuring OD260 and
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280 nm and confirmed by amplification with universal primers fD1 and rD1 previously
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described. The concentration of DNA was adjusted to 10 ng/µl and serially diluted in
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triplicate by using ultrapure sterile deionized water. Each DNA extract corresponding to one
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dilution level was tested for PCR amplification in duplicate, so that at least six PCR replicates
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were obtained for each concentration. The lowest amount of DNA with positive amplification
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in all replicates was chosen as the sensitivity limit.
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2.7. 16S rRNA sequencing
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The DNA extracted from 79 representative wild strains isolated from naturally contaminated
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milk powders was used to perform 16S rRNA amplification, according the conditions
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previously described. PCR products were verified by agarose (1% w/v) gel electrophoresis at
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7 V cm-1 for 2 h and sent to Fasteris SA (Plan-les Ouates, Genève, Switzerland) for their
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purification and sequencing. Research for DNA similarity was performed within the Gene 8
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Bank of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) by
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using the Basic Local Alignment Search Tool. The Gene Bank accession numbers of the
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sequences are reported in Table 2.
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2.8. Multiplex-PCR assay for the simultaneous identification of G. stearothermophilus and A.
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flavithermus
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A Multiplex-PCR assay was developed by using the two primer pairs, Fits2/Rits2 and Anx-
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RpoB5f/Anx-RpoB1bisr, to simultaneously identify G. stearothermophilus and A.
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flavithermus species. Each PCR mixture (final volume, 50 µl) was prepared as previously
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described and each primer was used at a concentration of 0.2 µΜ. PCR conditions were
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exactly the same applied for species-specific identification of G. stearothermophilus and A.
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flavithermus. The presence of PCR products was verified by agarose (2% w/v) gel
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electrophoresis at 7 V/cm for 2 h. DNA extracted from G. stearothermophilus DSM22 and A.
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flavithermus DSM2641 strains were used as control for the species-specific identification.
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3. RESULTS
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3.1. Validation of Geobacillus stearothermophilus PCR assay
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Species-specific PCR assay described by Prevost et al. (2010) was deeply modified to
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improve its specificity. By applying the PCR conditions described in the article (temperatures,
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time, number of cycles), several Geobacillus reference strains as well as A. flavithermus
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DSM2641T showed non-specific PCR products (Figure 2, Panel A) and, in particular, the
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strain G. thermodenitrificans DSM465T showed a band with the same molecular weight of the
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target species G. stearothermophilus (302 bp).
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Increasing the annealing temperature from 65°C to 69°C and shortening the PCR program, the
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PCR assay became highly specific for G. stearothermophilus species identification (Figure 2,
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Panel B). By applying this PCR program to the DNA extracted from 53 DSMZ reference
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strains, the expected 302 bp amplicon was revealed only when the DNA from the 5 DSMZ G.
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stearothermophilus strains was used (Table 1).
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3.2. Validation of Anoxybacillus flavithermus PCR assay
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The primer set Anx-RpoB5f/Anx-RpoB1bisr, designed on the basis of the rpoB gene
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sequences of 17 Anoxybacillus species available in the GeneBank nucleotide database, and the
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applied PCR conditions allowed a species-specific annealing only when the DNA extracted
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from A. flavithermus species was used (Table 1).
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The PCR assay exclusivity was tested with the DNA extracted from 53 DSMZ reference
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strains listed in table 1; the expected 142 bp amplicon was revealed only when the DNA from
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the two DSMZ A. flavithermus subsp. flavithermus strains was used (Table 1). No PCR
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amplification products were detected from the other reference strains (data not shown).
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3.3. Sensitivity of species-specific PCR assay
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The sensitivity of the PCR assays was determined by using the two primer sets Fits2/Rits2
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and Anx-RpoB5f/Anx-RpoB1bisr (Figure 3) as well as the universal fD1/rD1 set (data not
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shown).
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The species-specific G. stearothermophilus assay was more sensitive than the species-specific
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A. flavithermus assay; the detection limit per reaction was 5 pg of DNA for the former and 50
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pg for the latter. Both species-specific detection limits were identical to those obtained by the
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16S rRNA PCR assay.
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3.4. G. stearothermophilus and A. flavithermus identification from bulk cells and isolated
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wild strains by species-specific PCR assays
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In order to verify the reliability of the developed species-specific PCR assays, 24 samples
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(Table 2) were collected worldwide from different factories and used in this study to isolate 10
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wild strains and to collect bulk cells resistant to high temperatures. In particular, a total of 99
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wild strains were isolated from the 24 milk powder samples, while 23 bulk cells were
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collected; since the number of colonies grown on NA plates was very low, the isolation of the
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wild strains was preferred and no bulk cell was collected from sample D.
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Table 2 shows the results obtained after PCR amplification of the total DNA extracted from
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99 wild strains with the primer sets Fits2/Rits2 and Anx-Rpo5f/Anx-RpoB1bisr. 55 strains
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(55.6%) showed positive results to the Fits2/Rits2 PCR amplification, while 16 strains
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(16.2%) showed positive results to the Anx-Rpo5f/Anx-RpoB1bisr PCR amplification.
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Within the first group, 40 out of 55 strains were submitted to 16S rRNA sequencing to
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confirm the identification obtained by the species-specific PCR assay; all of them were
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identified as G. stearothermophilus. Within the second group, all the strains were submitted to
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16S rRNA sequencing and identified as belonging to the A. flavithermus species.
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The 55 G. stearothermophilus strains were isolated from 19 out of 24 milk powder samples
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(79.2%), while the 16 A. flavithermus strains were isolated from 6 out of 24 samples (25%).
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Moreover, both bacterial species were isolated from 4 milk powders (F, G, H and U). No
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target strains were isolated from 3 samples: Q, R and V.
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Three strains (Q3, R1 and V1) isolated from samples Q, R and V, respectively, were
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submitted to 16S rRNA sequencing to verify the trueness of the negative results obtained by
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the species-specific PCR assays; they were identified as Bacillus smithii, Brevibacillus
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thermoruber and Aeribacillus pallidus, respectively. As well, the 5 strains N4, O1, S1, T5 and
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U5, isolated from samples contaminated by G. stearothermophilus and/or A. flavithermus and
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giving negative results by species-specific PCR assays, were also identified by 16S rRNA
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sequencing as non-target species (Table 2).
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The results obtained after PCR amplification of the total DNA extracted from 23 bulk cells
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with the primer sets Fits2/Rits2 and Anx-Rpo5f/Anx-RpoB1bisr are reported in table 2. G.
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stearothermophilus was identified in the majority of samples (18 out of 23), while A.
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flavithermus was identified in 6 out of 23 bulk cells. As previously stated by analyzing the
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results of the species-specific identification of the wild strains, both bacterial species were
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isolated from 4 milk powders (F, G, H and U), while no target species was isolated from the 3
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samples Q, R and V.
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3.5. Multiplex-PCR assay for the simultaneous identification of G. stearothermophilus and A.
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flavithermus from bulk cells and isolated wild strains
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To simultaneously identify the two target species of the present study and shorten the analysis
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time, a Multiplex-PCR assay was developed. The Fits2/Rits2 and Anx-Rpo5f/Anx-RpoB1bisr
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primer sets and the PCR mixture previously described were used. Table 2 shows the results
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obtained by using the Multiplex-PCR assay with the DNA extracted from 99 wild strains and
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from 23 bulk cells; in particular, the specific PCR profiles obtained from each bulk cell are
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presented in Figure 4.
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The Multiplex-PCR assay successfully allowed the simultaneous detection and identification
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of the species G. stearothermophilus and A. flavithermus. The presence of both species in the
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same bulk cell sample did not interfere with the identification process as demonstrated by the
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double-band PCR profiles obtained for samples F, G, H (Panel A) and U (Panel C).
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Comparing the results obtained by applying the Multiplex-PCR assay and the single species-
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specific PCR assays, the bacterial identifications were always confirmed. The strains, not
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identified as belonging to the species G. stearothermophilus or A. flavithermus, and the bulk
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cells where the two target species were never detected, gave negative results by using the
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Multiplex-PCR assay.
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4. DISCUSSION
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G. stearothermophilus and A. flavithermus are the two main spore-forming species
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representing a concern for dairy industry. These obligate thermophilic bacilli can easily grow 12
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at elevated temperatures, can form biofilm and exhibit undesirable enzymatic activities
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leading to serious product defects (Chopra and Mathur, 1984; Ronimus et al., 2003; Chen et
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al., 2004; Scott et al., 2007) and health issues (Powlson et al., 2008).
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The ability to rapidly detect and identify these two target bacterial species would represent an
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economic advantage as well as a hygienic warranty for the consumers.
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This study was focused on the improvement and development of two PCR assays for the
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species-specific identification of G. stearothermophilus or A. flavithermus and, subsequently,
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on the development of a Multiplex-PCR assay for the simultaneous detection and
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identification of both species. Even if the preliminary enrichment/isolation step included in
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the methods increased the time of the analyses, it allowed the only detection of live cells,
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those really able to spoil UHT products, avoiding false positive or false negative results. The
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PCR assays were based on a very simple DNA extraction methodology and a rapid PCR
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protocol, without any extension step. These characteristics might allow the implementation of
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the identification procedure in dairy factories and its spread in laboratories where people
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without specific molecular biology skills are employed.
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When the species-specific primers designed by Prevost et al. (2010) for the identification of
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G. stearothermophilus and the related PCR conditions were applied, serious aspecificity
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problems were encountered. The PCR conditions were deeply modified; the short PCR
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procedure developed in this study resulted to be more specific, providing the expected PCR
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amplicon only when the DNA of the target species was used. The methodology significantly
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reduced the time of the analysis and it was successfully validated by using the DNA extracted
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from reference Geobacillus spp. strains and related spore-forming bacterial species.
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Flint et al. (2001) described a couple of primers designed on the basis of the 16S rRNA gene
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sequences for the species-specific identification of A. flavithermus. These primers were
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discarded for two main reasons. When the study was performed, A. flavithermus was still
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classified as Bacillus flavothermus and the primer sets were designed by comparing the 16S
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rRNA gene sequences of Bacillus related species. Additionally, Inan et al. (2011) stated that a
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molecular characterization of Anoxybacillus species is more discriminating and reliable by
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using rpoB gene sequences than the 16S rRNA gene sequences.
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The newly designed rpoB-based primer set was very specific; the expected PCR product was
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obtained by using the DNA belonging to the target A. flavithermus species, even when highly
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related Anoxybacillus spp. species, such as A. kestanbolensis, A. tengchongensis or A.
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eryuanensis (Inan et al., 2011) were analyzed.
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The species-specific G. stearothermophilus and A. flavithermus PCR assays showed a
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different sensitivity, with a limit of detection of 5 and 50 pg of DNA/reaction, respectively.
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The genome size of G. stearothermophilus is not yet defined, while the complete genome
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sequence of A. flavithermus strain WK1 is already available. It consists of a single
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chromosome of 2.846.746 bp (Saw et al., 2008); considering an average weight of 650 Da per
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base pair, the whole A. flavithermus WK1 genome is almost 1.850.385 KDa, equivalent to
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3.073x10-3 pg (1 CFU). 50 pg correspond to 1.63x104 CFU; this value represents the limit of
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detection of A. flavithermus in term of CFU/reaction of the PCR assay. However, since the
335
PCR assay include an enrichment/isolation step, high sensitivity does not represent a
336
fundamental prerequisite.
337
In the present study G. stearothermophilus and A. flavithermus were the most common spore-
338
forming bacilli contaminating UHT products, independently from their origin, as also stated
339
by Rueckert et al. (2004). The results showed that G. stearothermophilus was more frequent
340
than A. flavithermus; the former was detected in almost 80% of the samples, while the latter in
341
25% of them. Other thermophilic bacilli were identified in some milk powders, such as
342
Aerobacillus pallidus, Bacillus coagulans, Bacillus smithii and Brevibacillus thermoruber.
343
Among them, Bacillus smithii was found in 4 out of 24 samples (16.7%). This species is a
344
facultative thermophile with optimal growth temperature from 40 to 55°C, able to tolerate
345
maximum of 55-60°C (Logan and De Vos, 2001).
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The two PCR assays developed in this study were fulfilling all the defined objectives and
347
never caused a misleading identification of the analyzed strains. Moreover, when G.
348
stearothermophilus and/or A. flavithermus were detected in the bulk cells collected from the
349
milk powder samples, their presence was always confirmed by the identification of the
350
strain(s) isolated from the same sample.
351
The combination of the two assays in a unique Multiplex-PCR allowed a significant reduction
352
of the analyses time. This assay enabled the simultaneous identification of both target species,
353
confirming the identification results provided by the single PCR methodologies applied to
354
bulk cell or wild strain DNA.
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5. CONCLUSIONS
357
The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the
358
PCR procedures developed in this study highlight the advantage of their application for the
359
specific detection and identification of G. stearothermophilus and A. flavithermus.
360
Dairy factories adopting these methodologies might have beneficial tools to evaluate the
361
quality of the raw materials, to identify possible sources of bacterial contamination, to
362
monitor the efficacy of the procedures adopted to clean the manufacturing surfaces, to
363
implement preventive and corrective actions to reach and maintain targeted hygiene measures
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at appropriate levels and, finally, to evaluate the hygienic conditions of the finished products.
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Crielly, E.M., Logan, N.A. & Anderton, A. (1994). Studies on the Bacillus flora of milk and milk products. Journal of Applied Bacteriology, 77, 256-263. Denny, C.B. (1981). Thermophilic organisms involved in food spoilage - introduction.
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Flint, S.H., Ward, L.J.H. & Walker, K.M.R. (2001). Functional grouping of thermophilic
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Gundogan, N. & Arik, M.T. (2004). Comparison of the protease activity of psychrotrophic
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Hornstra, L.M., Ter Beek, A., Smelt, J.P., Kallemeijn, W.W. & Brul, S. (2009). On the origin
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Inan, K., Bektas, Y., Canakci, S. & Belduz, A.O. (2011). Use of rpoB sequences and rep-PCR
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Jay, J.M., Loessner, M.J. & Golden, D.A. (2005). Modern Food Microbiology. In Jay, J.M.,
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Logan, N.A. & De Vos, P. (2001). Bergey’s Manual of Systematic Bacteriology: The
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D.J., Krieg, W.R., Staley, J.T. (Eds.), Genus I. Bacillaceae, (pp. 21-128). New York:
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Postollec, F., Mathot, A.G., Bernard, M., Divanac’h, M.L., Pavan, S. & Sohier, D. (2012).
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Powlson, D.S., Addiscott, T.M., Benjamin, N., Cassman, K.G., de Kok, T.M., van Grinsven,
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H., L’Hirondel, J.L., Avery A.A. & van Kessel, C. (2008). When does the nitrate become a
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risk for humans? Journal of Environmental Quality, 37 (2), 291-295. Prevost, S., Andre, S. & Remize, F. (2010). PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage. Current Microbiology, 61, 525-533.
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Ronimus, R. S., Parker, L. E., Turner, N., Poudel, S., Ruckert, A., & Morgan, H. W. (2003).
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A RAPD-based comparison of thermophilic bacilli from milk powders. International
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Journal of Food Microbiology, 85 (1-2), 45-61. Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2004). A RAPD-based survey of thermophilic
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bacilli in milk powders from different countries. International Journal of Food
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Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2005a). Rapid differentiation and enumeration
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of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders
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Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2005b). Development of a rapid detection and
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enumeration method for thermophilic bacilli in milk powders. Journal of Microbiological
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Methods, 60, 155-167.
Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2006). Development of a real-time PCR assay
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targeting the sporulation gene, spo0A, for the enumeration of thermophilic bacilli in milk
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powder. Food Microbiology, 23, 220-230.
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Saw, J.H., Mountain, B.W., Feng, L., Omelchenko, M.V., Hou, S., Saito, J.A., Stott, M.B., Li,
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D., Zhao, G., Wu, J., Galperin, M.Y., Koonin, E.V., Makarova, K.S., Wolf, Y.I., Rigden,
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D.J., Dunfield, P.F., Wang, L. & Alam, M. (2008). Encapsulated in silica: genome,
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proteome and physiology of thermophic bacterium A. flavithermus WK1. Genome Biology, 9 (11), R161.
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Scott, S. A., Brooks, J. D., Rakonjac, J., Walker, K. M. R., & Flint, S. H. (2007). The
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formation of thermophilic spores during the manufacture of whole milk powder.
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International Journal of Dairy Technology, 60 (2), 109-117.
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Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994). CLUSTAL W: improving the
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sensitivity of progressive multiple sequence alignment through sequence weighting, 18
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position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22,
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4673-4680.
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phylogenetic study. Journal of Bacteriology, 173 (2), 697–703.
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Weisburg WG, Barns SM, Pelletier DA et al. (1991). 16S ribosomal DNA amplification for
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Table 1 Strains of the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) collection used in this study and evaluation of species-specificity of the PCR primers. Species-specific PCR with primer pair: Anx-RpoB5f/ Anx-RpoB1bisr
+ + + + + -
+ + -
RI PT
15939T 17956T 15730T 15866T 23212T 2641T 21510 23293 14988T 19169T 12423T 17127T 22626T 23211T 16325T 17141T 17075T 7T 31 T 1T 12T 13T 27T 10599T 10T 14992T 13864T 795T 4928T 12041T 16016T 18751T 7263T 22T 297 456 1550 2027 13552T 16325T 730T 465T 2542T 5366T 14590T 23175T 521T 1974T 15220T 24T 14349T 16487T 7060T
Fits2/Rits2
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Anoxybacillus amylolyticus Anoxybacillus bogrovensis Anoxybacillus caldiproteoliticus Anoxybacillus contaminans Anoxybacillus eryuanensis Anoxybacillus flavithermus subsp. flavithermus Anoxybacillus flavithermus subsp. flavithermus Anoxybacillus flavithermus subsp. yunnanensis Anoxybacillus kamchatkensis Anoxybacillus mongoliensis Anoxybacillus pushchinoensis Anoxybacillus rupiensis Anoxybacillus salavatliensis Anoxybacillus tengchongensis Anoxybacillus tepidamans Anoxybacillus thermarum Anoxybacillus voinovskiensis Bacillus amyloliquefaciens subsp. amyloliquefaciens Bacillus cereus Bacillus coagulans Bacillus firmus Bacillus licheniformis Bacillus pumilus Bacillus sporothermodurans Bacillus subtilis subsp. subtilis Clostridium novyi Clostridium saccharobutylicum Clostridium sporogenes Clostridium thermobutyricum Geobacillus caldoxylosilyticus Geobacillus debilis Geobacillus galactosidasius Geobacillus kaustophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus subterraneus Geobacillus tepidamans Geobacillus thermocatenulatus Geobacillus thermodenitrificans Geobacillus thermoglucosidasicus Geobacillus thermoleovorans Geobacillus toebii Geobacillus uzenensis Moorella thermoacetica Moorella thermoautotrophica Paenibacillus graminis Paenibacillus macerans Paenibacillus turicensis Thermoanaerobacterium aciditolerans Thermoanaerobacterium saccharolyticum
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
DSM strain
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Species
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Table 2 Origin of the IF milk powder samples and molecular identification of wild strains and bulk cells analyzed in this study.
C
Trinidad and Tobago
D
Trinidad and Tobago
E
Trinidad and Tobago
F
China
G
Pakistan
H
Pakistan
+ + + + + + + + + + + + + + + + + + + + + + + + + +
RI PT
Trinidad and Tobago
Fits2/Rits2
Anx-RpoB5f/ Anx-RpoB1bisr + + + + + + + + + + + + + +
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B
Wild strain A1 Wild strain A2 Bulk A Wild strain B1 Wild strain B2 Bulk B Wild strain C1 Wild strain C2 Wild strain C3 Wild strain C4 Bulk C Wild strain D1 Wild strain D2 Wild strain D3 Wild strain D4 Wild strain E1 Wild strain E2 Wild strain E3 Bulk E Wild strain F1 Wild strain F2 Wild strain F3 Wild strain F4 Wild strain F5 Bulk F Wild strain G1 Wild strain G2 Wild strain G3 Wild strain G4 Wild strain G5 Bulk G Wild strain H1 Wild strain H2 Wild strain H3 Wild strain H4 Wild strain H5 Bulk H
Multiplex-PCR assay
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Species-specific PCR with primer pair: Anx-RpoB5f/ Fits2/Rits2 Anx-RpoB1bisr + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
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DNA from:
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Origin
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16S rRNA sequencing
Accession n°
G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 100% G. stearothermophilus 100% G. stearothermophilus 100% G. stearothermophilus 100% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% G. stearothermophilus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% -
NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR026516 NR026516 NR040794 NR026516 NR026516 NR040794 NR040794 NR026516 NR026516 NR026516 NR026516 NR040794 NR026516 NR026516 NR026516 -
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L
Germany
M
N
O
P
Dominica Republic
Vietnam
Philippines
Morocco
RI PT
Trinidad and Tobago
-
SC
K
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
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Indonesia
-
TE D
J
EP
Trinidad and Tobago
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
AC C
I
Wild strain I1 Wild strain I2 Wild strain I3 Wild strain I4 Wild strain I5 Bulk I Wild strain J1 Wild strain J2 Bulk J Wild strain K1 Wild strain K2 Bulk K Wild strain L1 Wild strain L2 Wild strain L3 Wild strain L4 Wild strain L5 Bulk L Wild strain M1 Wild strain M2 Wild strain M3 Wild strain M4 Wild strain M5 Wild strain M6 Bulk M Wild strain N1 Wild strain N2 Wild strain N3 Wild strain N4 Wild strain N5 Wild strain N6 Bulk N Wild strain O1 Wild strain O2 Wild strain O3 Wild strain O4 Wild strain O5 Bulk O Wild strain P1 Wild strain P2 Wild strain P3 Wild strain P4 Wild strain P5
G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Not sequenced Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Aeribacillus pallidus 97% Not sequenced Not sequenced Bacillus smithii 99% Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Not sequenced Not sequenced
NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 FN428649
FN428653
KC119567
GQ293457 FN428649
FN428694
ACCEPTED MANUSCRIPT
U
V
Sri Lanka
Mexico
Indonesia
W
Indonesia
X
Pakistan
Not sequenced Not sequenced Bacillus smithii 99% Not sequenced Not sequenced Brevibacillus thermoruber 99% Not sequenced Not sequenced Not sequenced Not sequenced Bacillus smithii 99% Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Not sequenced G. stearothermophilus 100% Not sequenced Not sequenced Bacillus coagulans 99% A. flavithermus 99% G. stearothermophilus 100% Not sequenced Not sequenced Bacillus smithii 99% Aeribacillus pallidus 97% Not sequenced Not sequenced Not sequenced A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% -
RI PT
+ + + + + + + +
SC
+ + + + + + + -
M AN U
T
Pakistan
TE D
S
Brazil
+ + + + + + + +
EP
R
Spain
+ + + + + + + -
AC C
Q
Bulk P Wild strain Q1 Wild strain Q2 Wild strain Q3 Wild strain Q4 Wild strain Q5 Bulk Q Wild strain R1 Wild strain R2 Wild strain R3 Wild strain R4 Wild strain R5 Bulk R Wild strain S1 Wild strain S2 Wild strain S3 Wild strain S4 Wild strain S5 Bulk S Wild strain T1 Wild strain T2 Wild strain T3 Wild strain T4 Wild strain T5 Bulk T Wild strain U1 Wild strain U2 Wild strain U3 Wild strain U4 Wild strain U5 Bulk U Wild strain V1 Wild strain V2 Wild strain V3 Wild strain V4 Bulk V Wild strain W1 Wild strain W2 Wild strain W3 Bulk W Wild strain X1 Bulk X
-
EU652724
AY196006
EU652724 FN428694
FN428653
AB682458 KC429571 FN428694
EU652724 KC119567
KC429571 KC429571 KC429571 KC429571 -
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…ACGC …ACGC …ATGC …ACGT …ACGT …ACGT …GCGT …ACGT …ACGT …ACGT …ACGT …ATGT …ATGT …ACGT …ACGT …ACGT …ACGT
167 188 GCGACTTGCAGAAGACGGCACA GCGACTTGCAGAAGACGGCACA CAGAATTGCTGAGGATGGAACA GCCAGTAGCAGAGGATGGCACG TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGCAACA TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGGAACA TCCAATTACGGAAGATGGAACG TCCGATTGCCGAAGATGGAACG TCCGATTGCCGAAGATGGAACG TCCGATTGCGGAAGATGGAACG TCCGATTGCGGAAGATGGGACG TCCGATTGCGGAAGATGGAACA ACCGCTTGCGGAGGACGGCACG
1 A. amylolyticus DSM15939 (JF279467.1) 2 A. contaminans DSM15866 (JF279478.1) 3 A. rupiensis DSM17127 (JF279471.1) 4 A. voinovskiensis NCIMB13956 (JF279483.1) 5 A. kamchatkensis DSM14988 (JF279479.1) 6 Anoxybacillus spp. DSM21706 (JQ397277.1) 7 A. thermarum DSM17141 (JF279472.1) 8 A. ayderensis NCIMB13972 (JF279476.1) 9 A. gonensis NCIMB13933 (JF279473.1) 10 A. salavatliensis DSM22626 (JQ397278.1) 11 A. pushchinoensis DSM12423 (JF279477.1) 12 A. eryuanensis KCTC13720 (JF279469.1) 13 A. mongoliensis DSM19169 (JQ397279.1) 14 A. tengchongensis KCTC13721 (JF279470.1) 15 A. flavithermus DSM2641 (JF279475.1) 16 A. kestanbolensis NCIMB13971 (JF279474.1) 17 A. bogrovensis DSM17956 (JF279468.1)
…TCGT …TCGT …TCGT …TTGT …TTGT …TTGT …TTGT …TTGT …TTGT …ATGT …TTGT …TCGT …TCGT …TCGT …TTGT …TCGT …TCGT
286 308 CTCTGCAGCGACAGCGTGTATC TTCTGCAGCGACAGCGTGTATC ATCAGCGGCGACTGCGTGTATT ATCAGCAGCAACAGCTTGTATC GTCGGTGGCGACAGCGTGCATT GTCGGTGGCGACAGCGTGCATT ATCGGTGGCGACAGCGTGCATT GTCGGTGGCGACAGCGTGCATT GTCGGTAGCGACAGCGTGCATT GACATTAGCGACAGATCGCATT GTCAGTTGCGACTGCATGCATT GTCCGTAGCGACGGCTTGCATT GTCCGTAGCGACGGCTTGCATT GTCCGTAGCGACAGCCTGCATT ATCGGTAGCCACAGCGTGTATC ATCCGTAGCTACAGCGTGTATC CTCGGCAGCGACAGCGTGCATC
TT… TT… TT… TT… TT… TT… TT… TT… TT…
M AN U
SC
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1 A. amylolyticus DSM15939 (JF279467.1) 2 A. contaminans DSM15866 (JF279478.1) 3 A. rupiensis DSM17127 (JF279471.1) 4 A. voinovskiensis NCIMB13956 (JF279483.1) 5 A. kamchatkensis DSM14988 (JF279479.1) 6 Anoxybacillus spp. DSM21706 (JQ397277.1) 7 A. thermarum DSM17141 (JF279472.1) 8 A. ayderensis NCIMB13972 (JF279476.1) 9 A. gonensis NCIMB13933 (JF279473.1) 10 A. salavatliensis DSM22626 (JQ397278.1) 11 A. pushchinoensis DSM12423 (JF279477.1) 12 A. eryuanensis KCTC13720 (JF279469.1) 13 A. mongoliensis DSM19169 (JQ397279.1) 14 A. tengchongensis KCTC13721 (JF279470.1) 15 A. flavithermus DSM2641 (JF279475.1) 16 A. kestanbolensis NCIMB13971 (JF279474.1) 17 A. bogrovensis DSM17956 (JF279468.1)
AT… TT… TT… TT… TT… TT… TT… TT…
CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC…
AC C
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Figure 1 - Sequence alignment of portions of rpoB gene sequences of A. flavithermus and other Anoxybacillus species. The accession numbers of the sequences used for the alignment are indicated. Variable sequences are highlighted in white. The sequences used for primer design are boxed.
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Figure 2 – Species-specific PCR assay by using the primer pair: Fits2/Rits2. Panel A: species-specific PCR assay by applying Prevost et al. (2010) PCR conditions (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, G. stearothermophilus DSM22T; lane 2, G. subterraneus DSM13552T; lane 3, G. caldoxylosilyticus DSM12041T; lane 4, G. thermodenitrificans DSM465T; lane 5, G. kaustophilus DSM7263T; lane 6, Clostridium thermobutyricum DSM4928T; lane 7, A. flavithermus DSM2641T; lane 8, Bacillus subtilis DSM10T; lane 9, negative control (deionized water); M, 1Kb Plus DNA Ladder. Panel B: species-specific PCR assay by applying PCR conditions developed in this study (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, G. stearothermophilus DSM22T; lane 2, G. subterraneus DSM13552T; lane 3, G. uzenensis DSM23175T; lane 4, G. caldoxylosilyticus DSM12041T; lane 5, G. thermoleovorans DSM5366T; lane 6, G. thermodenitrificans DSM465T; lane 7, G. kaustophilus DSM7263T; lane 8, Clostridium thermobutyricum DSM4928T; lane 9, Thermoanaerobacterium aciditolerans DSM16487T; lane 10, A. flavithermus DSM2641T; lane 11, Bacillus cereus DSM31T; lane 12, Bacillus subtilis DSM10T; M, 1Kb Plus DNA Ladder.
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Figure 3 – Sensitivity of the PCR assays. Upper part: Species-specific PCR assay with G. stearothermophilus DSM22T DNA as template; from left to the right: M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, 10 ng of DNA; lane 2, 5 ng of DNA; lane 3, 500 pg of DNA; lane 4, 50 pg of DNA; lane 5, 5 pg of DNA; lane 6, 0.5 pg of DNA; lane 7, 50 fg of DNA; lane 8, negative control; M, 1Kb Plus DNA Ladder (Invitrogen). Lower part: Species-specific PCR assay with A. flavithermus DSM2641T DNA as template (in duplicate); from left to the right: M, 1Kb Plus DNA Ladder (Invitrogen); lane 1-2, 10 ng of DNA; lane 3-4, 5 ng of DNA; lane 5-6, 500 pg of DNA; lane 7-8, 50 pg of DNA; lane 9-10, 5 pg of DNA; lane 11-12, 0.5 pg of DNA; M, 1Kb Plus DNA Ladder (Invitrogen).
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Figure 4 – Multiplex-PCR profiles obtained with bulk cells collected from 23 milk powder samples. Panel A (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, bulk cell A; lane 2, bulk cell B; lane 3, bulk cell C; lane 4, bulk cell E; lane 5, bulk cell F; lane 6, bulk cell G; lane 7, bulk cell H; lane 8, bulk cell I; lane 9, bulk cell J; lane 10, bulk cell K; lane 11, bulk cell L; lane 12, G. stearothermophilus DSM22T; lane 13, A. flavithermus DSM2641T; lane 14, negative control; M, 1Kb Plus DNA Ladder (Invitrogen). Panel B (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, bulk cell M; lane 2, bulk cell N; lane 3, bulk cell O; lane 4, bulk cell P; lane 5, bulk cell Q; lane 6, G. stearothermophilus DSM22T; lane 7, A. flavithermus DSM2641T; lane 8, negative control. Panel C (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, bulk cell R; lane 2, bulk cell
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S; lane 3, bulk cell T; lane 4, bulk cell U; lane 5, bulk cell V; lane 6, bulk cell W; lane 7, bulk cell X; lane 8, G. stearothermophilus DSM22T; lane 9, A. flavithermus DSM2641T; lane 10, negative control.
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HIGHLIGHTS G. stearothermophilus and A. flavithermus identifications were achieved by PCR assays
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Primers targeting ITS 16S-23S rRNA region and rpoB gene sequence were employed
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After testing their specificity, they were combined in a Multiplex-PCR assay
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The simultaneous detection of the two target species was successfully achieved
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Rapidity (~ 4h from DNA isolation to results) of the assays emphasize their advantage
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S0740-0020(14)00095-1
DOI:
10.1016/j.fm.2014.05.002
Reference:
YFMIC 2161
To appear in:
Food Microbiology
Received Date: 20 August 2013 Revised Date:
29 April 2014
Accepted Date: 2 May 2014
Please cite this article as: Pennacchia, C., Breeuwer, P., Meyer, R., Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus, Food Microbiology (2014), doi: 10.1016/j.fm.2014.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development of a Multiplex-PCR assay for the rapid identification of Geobacillus
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stearothermophilus and Anoxybacillus flavithermus
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Carmela Pennacchia*,a, Pieter Breeuwerb and Rolf Meyera
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a
Nestlé Product Technology Center (PTC), NESTEC Ltd, Nestlé Strasse 3, 3510 Konolfingen, Switzerland.
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b
Nestlé Nutrition, NESTEC Ltd, Avenue Reller 22, 1800 Vevey, Switzerland.
7 *Corresponding author: Tel.: +41 (0)31 790 1815; fax: +41 (0) 31 790 1552
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E-mail address: [email protected]
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ABSTRACT
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The presence of thermophilic bacilli in dairy products is indicator of poor hygiene. Their
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rapid detection and identification is fundamental to improve the industrial reactivity in the
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implementation of corrective and preventive actions.
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In this study a rapid and reliable identification of Geobacillus stearothermophilus and
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Anoxybacillus flavithermus was achieved by species-specific PCR assays. Two primer sets,
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targeting the ITS 16S-23S rRNA region and the rpoB gene sequence of the target species
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respectively, were employed. Species-specificity of both primer sets was evaluated by using
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53 reference strains of DSMZ collection; among them, 13 species of the genus Geobacillus
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and 15 of the genus Anoxybacillus were represented. Moreover, 99 wild strains and 23 bulk
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cells collected from 24 infant formula powders gathered from several countries worldwide
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were included in the analyses. Both primer sets were highly specific and the expected PCR
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fragments were obtained only when DNA from G. stearothermophilus or A. flavithermus was
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used. After testing their specificity, they were combined in a Multiplex-PCR assay for the
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simultaneous identification of the two target species. The specificity of the Multiplex-PCR
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was evaluated by using both wild strains and bulk cells. Every analysis confirmed the reliable
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identification results provided by the single species-specific PCR methodology.
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The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the
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PCR procedures developed in this study highlight the advantage of their application for the
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specific detection and identification of the thermophilic species G. stearothermophilus and A.
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flavithermus.
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Keywords: Geobacillus stearothermophilus, Anoxybacillus flavithermus, thermophilic
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bacilli, milk powder, PCR.
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1. INTRODUCTION
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Thermal processes based on high temperatures are commonly used in food industries to
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guarantee that food products remain stable for long period at ambient temperatures (Prevost et
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al., 2010). However, the thermal treatments are not always sufficient to inactivate all spore-
41
forming bacteria, especially those that are highly heat-resistant, but non-pathogenic (Hornstra
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et al., 2009). Among them, the thermophilic bacilli were reported to be important
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contaminants in milk powders (Ronimus et al., 2003; Ruckert at al., 2004, Scott et al., 2007),
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canned foods and dairy products (Denny, 1981; Jay et al., 2005; Scott et al., 2007; Prevost et
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al., 2010).
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In the dairy industry, the thermophilic bacilli can be divided in two main groups: facultative
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thermophiles (also known as thermotolerant microorganisms) and obligate thermophiles. The
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obligate thermophiles grow at elevated temperatures (approximately 48-60°C) and mainly
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include the two species: Geobacillus (G.) stearothermophilus and Anoxybacillus (A.)
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flavithermus (Flint et al., 2001; Ronimus et al., 2003; Scott et al., 2007; Burgess et al., 2010).
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Both species exhibit a fast growth rate and tend to form biofilm on the stainless steel surfaces
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of processing equipment (Scott et al., 2007).
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The presence of the thermophilic bacilli in dairy products is indicator of poor hygiene; high
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counts are unacceptable, since they can lead to product defects caused by the production of
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heat-stable enzymes, such as proteinases and lipases, and acids capable to spoil the final
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product (Chopra and Mathur, 1984; Cosentino et al., 1997; Chen et al., 2004; Gundogan and
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Arik, 2004).
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Identification of the bacteria capable to contaminate milk powders and possibly cause their
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spoilage can help in implementing corrective and preventive actions, in particular at level of
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the manufacturing process steps before heat treatment. Molecular methods able to rapidly
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detect and identify thermophilic contaminants are fundamental to improve the industrial
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reactivity (Prevost et al., 2010).
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In the last few years several efforts were directed toward the development of PCR-based
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methods for the investigation of the thermophilic bacilli contamination in milk powders, but
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most of the studies were focused on their total enumeration by quantitative real-time PCR,
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without distinguishing among the different species. Moreover, some identification methods
67
were too expensive or required a lot of expertise to be easily implemented in the routine
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analysis scheme of the industrial laboratories (Flint et al., 2001; Ronimus et al., 2003;
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Rueckert et al., 2005 a-b; Rueckert et al., 2006; Prevost et al., 2010; Postollec et al., 2012).
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The main objective of this study was to develop rapid and easy PCR-based assays for the
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detection and identification of the thermophilic species G. stearothermophilus and A.
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flavithermus. Both species detection was based on activation, germination and outgrowth of
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the spores on agar plates followed by PCR. This step was preferred to a direct PCR detection
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from milk powder samples to prevent false-negative reactions due to inhibitory substances
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present in the milks like calcium ions (Bickley et al., 1996) or proteinases (Powell et al.,
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1994) as well as false-positive reactions due to the free DNA of dead bacteria.
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The G. stearothermophilus species-specific PCR assay described by Prevost et al. (2010) was
78
deeply modified. A new primer set for the species-specific identification of A. flavithermus
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was designed by the alignment and comparison of the rpoB gene sequences of Anoxybacillus
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species. Specificity of both PCR assays was validated by testing reference strains and by
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analysing the 16S rRNA sequence of a representative number of wild strains isolated from
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naturally contaminated milk powder samples collected worldwide. The species-specific PCR
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assays were also tested to detect and identify the two target species in the bulk cells collected
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from the same milk powder samples.
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After fulfilling the validation process, the two PCR assays were combined in a unique reliable
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Multiplex-PCR assay, capable to further halve the analysis time and cost.
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2. MATERIALS AND METHODS
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2.1. Bacterial reference strains and growth conditions
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This study involved 53 reference strains from DSMZ collection (Table 1), representing 15
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different species of the genus Anoxybacillus, eight different species of the genus Bacillus,
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four different species of the genus Clostridium, 13 different species of the genus Geobacillus,
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two different species of the genus Moorella, three different species of the genus Paenibacillus
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and two different species of the genus Thermoanaerobacterium.
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With the exception of G. stearothermophilus strains 297, 456, 1550 and 2027, all the
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reference strains were purchased as ready-to-use DNA from DSMZ.
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Working cultures of G. stearothermophilus strains were purchased as lyophilized cultures and
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were grown on Nutrient Agar (NA; Oxoid Ltd., Basingstoke, Hampshire, UK) at 60°C in
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aerobic conditions for 48 h.
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2.2. Isolation of wild strains from infant formula (IF) milk powders
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24 IF milk powder samples (Table 2) were collected worldwide from different factories and
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used to isolate wild strains, naturally contaminating IF powders and resistant to high
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temperature.
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Ten grams of each sample were transferred into a sterile Stomacher bag, reconstituted with 90
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ml of Tryptone Sodium Chloride broth + antifoam (TS+ broth; Tryptone, Oxoid; Sodium
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Chloride, Merck; Silicon antifoam, Sigma) with the addition of 2 g/l of soluble starch (Merck)
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and treated with a Stomacher machine for 30 sec. To induce spore outgrowth, milk
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suspensions were heat-treated at 100°C for 30 minutes in an oil bath and then incubated at
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60°C for 48 h to promote the growth of thermophilic bacilli. After the incubation, serial
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decimal dilutions were prepared by using Maximum Recovery Diluent (MRD; Oxoid) and 0.1
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ml were spread-plated on NA plates in duplicate. Plates were incubated at 60°C for 48 h
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under aerobic conditions.
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After the incubation, the first plate from each sample showing a number of colonies from 20
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to 200 CFU was used to purify and isolate 2-5 colonies showing different morphology, while
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the duplicate plate was used for the collection of bulk cells. The list of the wild strains
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isolated from each milk powder sample is reported in Table 2.
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2.3. Collection of bulk cells
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The cultivable high temperature-resistant microbiota of 23 IF milk powder samples were
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collected from the duplicate NA plates showing 20-200 CFU (Table 2). All colonies present
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on the plates were collected by gently scraping of the surface with a sterile spatula, and each
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was resuspended in 2 ml of MRD and collected in a vial (“bulk cells”). After centrifugation at
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12.000 rpm for 10 min, each pellet was used for DNA extraction.
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For each IF sample, two replicates were prepared; the bulk cells collection from each replicate
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was conducted in duplicate.
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2.4. DNA extraction
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DNA extraction was carried out from the 4 G. stearothermophilus DSM strains 297, 456,
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1550 and 2027, from the pellet of bulk cells and from purified wild strains by using the
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InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA), following the procedure described
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by the supplier. DNA extraction from each bulk cell and from each wild strain was performed
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in triplicate. After extraction, DNA was quantified by using the NanoDrop Lite
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Spectrophotometer (ThermoScientific, Wilmington, DE, USA), standardized at 25 ng/µl by
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using ultra-pure sterile deionized water and 50 ng of DNA were used for PCR amplification.
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2.5. PCR conditions
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To verify that the extracted DNA could be amplified, each wild strain’s DNA was submitted
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to the 16S rRNA amplification by using the universal primers for eubacteria fD1 (5’-AGA 6
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GTT TGA TCH TGG CTC AG-3’) and rD1 (5’-GGM TAC CTT GTT ACG AYT TC-3’)
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(Escherichia coli positions 8–17 and 1540–1524, respectively) described by Weisburg et al.
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(1991). Each DNA was tested for PCR amplification in duplicate. Each PCR mixture (final
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volume, 50 µl) contained 50 ng of template DNA, each primer at a concentration of 0.2
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µΜ, each deoxynucleoside triphosphate at a concentration of 0.25 mM, 2.5 mM MgCl2, 5 µl
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of 10X PCR buffer (Invitrogen, Life Technologies Europe B.V.) and 2.5 U of Taq
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polymerase (Invitrogen). DNA amplification was performed by using the thermocycler
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GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies Europe B.V.). PCR
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conditions consisted of 1 cycle at 94°C for 3 min to denaturate DNA followed by 30 cycles (1
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min at 94°C, 45 sec at 54°C, 2 min at 72°C) plus one additional cycle at 72°C for 7 min as a
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final chain elongation. The presence of PCR products was verified by agarose (1% w/v) gel
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electrophoresis at 7 V/cm for 2 h.
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Species-specific PCR for G. stearothermophilus identification was conducted by using the
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primers Fits2 (5’-GGG GAA GCG CCG CGT TCG G-3’) and Rits2 (5’-GTG CAA GCA
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CCC TTG CAG GCG AAG A-3’), already described by Prevost et al. (2010) and targeting
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the ITS 16S-23S rRNA region. Each PCR mixture (final volume, 50 µl) was prepared as
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described above and PCR conditions were deeply modified, since those described by Prevost
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et al. (2010) were not highly specific for the G. stearothermophilus species only. In particular,
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PCR conditions were shortened by applying 1 cycle at 94°C for 3 min, to denaturate DNA,
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followed by 25 cycles (15 sec at 94°C, 30 sec at 69°C), without elongation step. Each DNA
159
was tested for PCR amplification in duplicate. The presence of the 302 bp PCR products was
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verified by agarose (2% w/v) gel electrophoresis at 7 V/cm for 2 h.
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Species-specific PCR for A. flavithermus identification was conducted by using the newly
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designed primers Anx-RpoB5f (5’- TCC GAT TGC GGA AGA TGG GAC G -3’) and Anx-
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RpoB1bisr (5’-GAT ACA CGC TGT GGC TAC CGA T-3’). The primers were designed on
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the basis of the comparison of the rpoB gene sequences of A. flavithermus and of the
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Anoxybacillus species listed in Table 1. Sequence alignment was performed by ClustalW
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program (Thompson et al., 1994). The alignment is showed in Figure 1, where the sequence
167
variability used for the species-specific design of the primers is highlighted. The primers were
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targeting the rpoB gene in positions 167-188 and 308-286, respectively and the expected size
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of the PCR product was of 142 bp. PCR mixture and PCR conditions applied were exactly the
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same applied for species-specific identification of G. stearothermophilus.
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2.6. Determination of the species-specific PCR sensitivities
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The sensitivity was determined for each of the two species-specific PCR primer pairs with
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DNA extracted from reference strains A. flavithermus DSM 2641 and G. stearothermophilus
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DSM22.
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The level and purity of DNA of the extracted DNA were evaluated by measuring OD260 and
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280 nm and confirmed by amplification with universal primers fD1 and rD1 previously
178
described. The concentration of DNA was adjusted to 10 ng/µl and serially diluted in
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triplicate by using ultrapure sterile deionized water. Each DNA extract corresponding to one
180
dilution level was tested for PCR amplification in duplicate, so that at least six PCR replicates
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were obtained for each concentration. The lowest amount of DNA with positive amplification
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in all replicates was chosen as the sensitivity limit.
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2.7. 16S rRNA sequencing
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The DNA extracted from 79 representative wild strains isolated from naturally contaminated
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milk powders was used to perform 16S rRNA amplification, according the conditions
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previously described. PCR products were verified by agarose (1% w/v) gel electrophoresis at
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7 V cm-1 for 2 h and sent to Fasteris SA (Plan-les Ouates, Genève, Switzerland) for their
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purification and sequencing. Research for DNA similarity was performed within the Gene 8
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Bank of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) by
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using the Basic Local Alignment Search Tool. The Gene Bank accession numbers of the
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sequences are reported in Table 2.
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2.8. Multiplex-PCR assay for the simultaneous identification of G. stearothermophilus and A.
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flavithermus
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A Multiplex-PCR assay was developed by using the two primer pairs, Fits2/Rits2 and Anx-
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RpoB5f/Anx-RpoB1bisr, to simultaneously identify G. stearothermophilus and A.
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flavithermus species. Each PCR mixture (final volume, 50 µl) was prepared as previously
199
described and each primer was used at a concentration of 0.2 µΜ. PCR conditions were
200
exactly the same applied for species-specific identification of G. stearothermophilus and A.
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flavithermus. The presence of PCR products was verified by agarose (2% w/v) gel
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electrophoresis at 7 V/cm for 2 h. DNA extracted from G. stearothermophilus DSM22 and A.
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flavithermus DSM2641 strains were used as control for the species-specific identification.
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3. RESULTS
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3.1. Validation of Geobacillus stearothermophilus PCR assay
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Species-specific PCR assay described by Prevost et al. (2010) was deeply modified to
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improve its specificity. By applying the PCR conditions described in the article (temperatures,
209
time, number of cycles), several Geobacillus reference strains as well as A. flavithermus
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DSM2641T showed non-specific PCR products (Figure 2, Panel A) and, in particular, the
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strain G. thermodenitrificans DSM465T showed a band with the same molecular weight of the
212
target species G. stearothermophilus (302 bp).
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Increasing the annealing temperature from 65°C to 69°C and shortening the PCR program, the
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PCR assay became highly specific for G. stearothermophilus species identification (Figure 2,
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Panel B). By applying this PCR program to the DNA extracted from 53 DSMZ reference
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strains, the expected 302 bp amplicon was revealed only when the DNA from the 5 DSMZ G.
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stearothermophilus strains was used (Table 1).
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3.2. Validation of Anoxybacillus flavithermus PCR assay
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The primer set Anx-RpoB5f/Anx-RpoB1bisr, designed on the basis of the rpoB gene
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sequences of 17 Anoxybacillus species available in the GeneBank nucleotide database, and the
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applied PCR conditions allowed a species-specific annealing only when the DNA extracted
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from A. flavithermus species was used (Table 1).
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The PCR assay exclusivity was tested with the DNA extracted from 53 DSMZ reference
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strains listed in table 1; the expected 142 bp amplicon was revealed only when the DNA from
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the two DSMZ A. flavithermus subsp. flavithermus strains was used (Table 1). No PCR
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amplification products were detected from the other reference strains (data not shown).
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3.3. Sensitivity of species-specific PCR assay
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The sensitivity of the PCR assays was determined by using the two primer sets Fits2/Rits2
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and Anx-RpoB5f/Anx-RpoB1bisr (Figure 3) as well as the universal fD1/rD1 set (data not
232
shown).
233
The species-specific G. stearothermophilus assay was more sensitive than the species-specific
234
A. flavithermus assay; the detection limit per reaction was 5 pg of DNA for the former and 50
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pg for the latter. Both species-specific detection limits were identical to those obtained by the
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16S rRNA PCR assay.
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3.4. G. stearothermophilus and A. flavithermus identification from bulk cells and isolated
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wild strains by species-specific PCR assays
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In order to verify the reliability of the developed species-specific PCR assays, 24 samples
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(Table 2) were collected worldwide from different factories and used in this study to isolate 10
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wild strains and to collect bulk cells resistant to high temperatures. In particular, a total of 99
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wild strains were isolated from the 24 milk powder samples, while 23 bulk cells were
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collected; since the number of colonies grown on NA plates was very low, the isolation of the
245
wild strains was preferred and no bulk cell was collected from sample D.
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Table 2 shows the results obtained after PCR amplification of the total DNA extracted from
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99 wild strains with the primer sets Fits2/Rits2 and Anx-Rpo5f/Anx-RpoB1bisr. 55 strains
248
(55.6%) showed positive results to the Fits2/Rits2 PCR amplification, while 16 strains
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(16.2%) showed positive results to the Anx-Rpo5f/Anx-RpoB1bisr PCR amplification.
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Within the first group, 40 out of 55 strains were submitted to 16S rRNA sequencing to
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confirm the identification obtained by the species-specific PCR assay; all of them were
252
identified as G. stearothermophilus. Within the second group, all the strains were submitted to
253
16S rRNA sequencing and identified as belonging to the A. flavithermus species.
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The 55 G. stearothermophilus strains were isolated from 19 out of 24 milk powder samples
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(79.2%), while the 16 A. flavithermus strains were isolated from 6 out of 24 samples (25%).
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Moreover, both bacterial species were isolated from 4 milk powders (F, G, H and U). No
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target strains were isolated from 3 samples: Q, R and V.
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Three strains (Q3, R1 and V1) isolated from samples Q, R and V, respectively, were
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submitted to 16S rRNA sequencing to verify the trueness of the negative results obtained by
260
the species-specific PCR assays; they were identified as Bacillus smithii, Brevibacillus
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thermoruber and Aeribacillus pallidus, respectively. As well, the 5 strains N4, O1, S1, T5 and
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U5, isolated from samples contaminated by G. stearothermophilus and/or A. flavithermus and
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giving negative results by species-specific PCR assays, were also identified by 16S rRNA
264
sequencing as non-target species (Table 2).
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The results obtained after PCR amplification of the total DNA extracted from 23 bulk cells
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with the primer sets Fits2/Rits2 and Anx-Rpo5f/Anx-RpoB1bisr are reported in table 2. G.
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stearothermophilus was identified in the majority of samples (18 out of 23), while A.
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flavithermus was identified in 6 out of 23 bulk cells. As previously stated by analyzing the
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results of the species-specific identification of the wild strains, both bacterial species were
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isolated from 4 milk powders (F, G, H and U), while no target species was isolated from the 3
271
samples Q, R and V.
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3.5. Multiplex-PCR assay for the simultaneous identification of G. stearothermophilus and A.
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flavithermus from bulk cells and isolated wild strains
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To simultaneously identify the two target species of the present study and shorten the analysis
276
time, a Multiplex-PCR assay was developed. The Fits2/Rits2 and Anx-Rpo5f/Anx-RpoB1bisr
277
primer sets and the PCR mixture previously described were used. Table 2 shows the results
278
obtained by using the Multiplex-PCR assay with the DNA extracted from 99 wild strains and
279
from 23 bulk cells; in particular, the specific PCR profiles obtained from each bulk cell are
280
presented in Figure 4.
281
The Multiplex-PCR assay successfully allowed the simultaneous detection and identification
282
of the species G. stearothermophilus and A. flavithermus. The presence of both species in the
283
same bulk cell sample did not interfere with the identification process as demonstrated by the
284
double-band PCR profiles obtained for samples F, G, H (Panel A) and U (Panel C).
285
Comparing the results obtained by applying the Multiplex-PCR assay and the single species-
286
specific PCR assays, the bacterial identifications were always confirmed. The strains, not
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identified as belonging to the species G. stearothermophilus or A. flavithermus, and the bulk
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cells where the two target species were never detected, gave negative results by using the
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Multiplex-PCR assay.
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4. DISCUSSION
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G. stearothermophilus and A. flavithermus are the two main spore-forming species
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representing a concern for dairy industry. These obligate thermophilic bacilli can easily grow 12
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at elevated temperatures, can form biofilm and exhibit undesirable enzymatic activities
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leading to serious product defects (Chopra and Mathur, 1984; Ronimus et al., 2003; Chen et
296
al., 2004; Scott et al., 2007) and health issues (Powlson et al., 2008).
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The ability to rapidly detect and identify these two target bacterial species would represent an
298
economic advantage as well as a hygienic warranty for the consumers.
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This study was focused on the improvement and development of two PCR assays for the
300
species-specific identification of G. stearothermophilus or A. flavithermus and, subsequently,
301
on the development of a Multiplex-PCR assay for the simultaneous detection and
302
identification of both species. Even if the preliminary enrichment/isolation step included in
303
the methods increased the time of the analyses, it allowed the only detection of live cells,
304
those really able to spoil UHT products, avoiding false positive or false negative results. The
305
PCR assays were based on a very simple DNA extraction methodology and a rapid PCR
306
protocol, without any extension step. These characteristics might allow the implementation of
307
the identification procedure in dairy factories and its spread in laboratories where people
308
without specific molecular biology skills are employed.
309
When the species-specific primers designed by Prevost et al. (2010) for the identification of
310
G. stearothermophilus and the related PCR conditions were applied, serious aspecificity
311
problems were encountered. The PCR conditions were deeply modified; the short PCR
312
procedure developed in this study resulted to be more specific, providing the expected PCR
313
amplicon only when the DNA of the target species was used. The methodology significantly
314
reduced the time of the analysis and it was successfully validated by using the DNA extracted
315
from reference Geobacillus spp. strains and related spore-forming bacterial species.
316
Flint et al. (2001) described a couple of primers designed on the basis of the 16S rRNA gene
317
sequences for the species-specific identification of A. flavithermus. These primers were
318
discarded for two main reasons. When the study was performed, A. flavithermus was still
319
classified as Bacillus flavothermus and the primer sets were designed by comparing the 16S
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rRNA gene sequences of Bacillus related species. Additionally, Inan et al. (2011) stated that a
321
molecular characterization of Anoxybacillus species is more discriminating and reliable by
322
using rpoB gene sequences than the 16S rRNA gene sequences.
323
The newly designed rpoB-based primer set was very specific; the expected PCR product was
324
obtained by using the DNA belonging to the target A. flavithermus species, even when highly
325
related Anoxybacillus spp. species, such as A. kestanbolensis, A. tengchongensis or A.
326
eryuanensis (Inan et al., 2011) were analyzed.
327
The species-specific G. stearothermophilus and A. flavithermus PCR assays showed a
328
different sensitivity, with a limit of detection of 5 and 50 pg of DNA/reaction, respectively.
329
The genome size of G. stearothermophilus is not yet defined, while the complete genome
330
sequence of A. flavithermus strain WK1 is already available. It consists of a single
331
chromosome of 2.846.746 bp (Saw et al., 2008); considering an average weight of 650 Da per
332
base pair, the whole A. flavithermus WK1 genome is almost 1.850.385 KDa, equivalent to
333
3.073x10-3 pg (1 CFU). 50 pg correspond to 1.63x104 CFU; this value represents the limit of
334
detection of A. flavithermus in term of CFU/reaction of the PCR assay. However, since the
335
PCR assay include an enrichment/isolation step, high sensitivity does not represent a
336
fundamental prerequisite.
337
In the present study G. stearothermophilus and A. flavithermus were the most common spore-
338
forming bacilli contaminating UHT products, independently from their origin, as also stated
339
by Rueckert et al. (2004). The results showed that G. stearothermophilus was more frequent
340
than A. flavithermus; the former was detected in almost 80% of the samples, while the latter in
341
25% of them. Other thermophilic bacilli were identified in some milk powders, such as
342
Aerobacillus pallidus, Bacillus coagulans, Bacillus smithii and Brevibacillus thermoruber.
343
Among them, Bacillus smithii was found in 4 out of 24 samples (16.7%). This species is a
344
facultative thermophile with optimal growth temperature from 40 to 55°C, able to tolerate
345
maximum of 55-60°C (Logan and De Vos, 2001).
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The two PCR assays developed in this study were fulfilling all the defined objectives and
347
never caused a misleading identification of the analyzed strains. Moreover, when G.
348
stearothermophilus and/or A. flavithermus were detected in the bulk cells collected from the
349
milk powder samples, their presence was always confirmed by the identification of the
350
strain(s) isolated from the same sample.
351
The combination of the two assays in a unique Multiplex-PCR allowed a significant reduction
352
of the analyses time. This assay enabled the simultaneous identification of both target species,
353
confirming the identification results provided by the single PCR methodologies applied to
354
bulk cell or wild strain DNA.
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5. CONCLUSIONS
357
The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the
358
PCR procedures developed in this study highlight the advantage of their application for the
359
specific detection and identification of G. stearothermophilus and A. flavithermus.
360
Dairy factories adopting these methodologies might have beneficial tools to evaluate the
361
quality of the raw materials, to identify possible sources of bacterial contamination, to
362
monitor the efficacy of the procedures adopted to clean the manufacturing surfaces, to
363
implement preventive and corrective actions to reach and maintain targeted hygiene measures
364
at appropriate levels and, finally, to evaluate the hygienic conditions of the finished products.
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Gundogan, N. & Arik, M.T. (2004). Comparison of the protease activity of psychrotrophic
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Hornstra, L.M., Ter Beek, A., Smelt, J.P., Kallemeijn, W.W. & Brul, S. (2009). On the origin
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Logan, N.A. & De Vos, P. (2001). Bergey’s Manual of Systematic Bacteriology: The
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D.J., Krieg, W.R., Staley, J.T. (Eds.), Genus I. Bacillaceae, (pp. 21-128). New York:
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Postollec, F., Mathot, A.G., Bernard, M., Divanac’h, M.L., Pavan, S. & Sohier, D. (2012).
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H., L’Hirondel, J.L., Avery A.A. & van Kessel, C. (2008). When does the nitrate become a
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risk for humans? Journal of Environmental Quality, 37 (2), 291-295. Prevost, S., Andre, S. & Remize, F. (2010). PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage. Current Microbiology, 61, 525-533.
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Ronimus, R. S., Parker, L. E., Turner, N., Poudel, S., Ruckert, A., & Morgan, H. W. (2003).
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A RAPD-based comparison of thermophilic bacilli from milk powders. International
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Journal of Food Microbiology, 85 (1-2), 45-61. Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2004). A RAPD-based survey of thermophilic
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bacilli in milk powders from different countries. International Journal of Food
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Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2005a). Rapid differentiation and enumeration
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of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders
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Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2005b). Development of a rapid detection and
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enumeration method for thermophilic bacilli in milk powders. Journal of Microbiological
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Methods, 60, 155-167.
Rueckert, A., Ronimus, R.S. & Morgan, H.W. (2006). Development of a real-time PCR assay
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targeting the sporulation gene, spo0A, for the enumeration of thermophilic bacilli in milk
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powder. Food Microbiology, 23, 220-230.
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Saw, J.H., Mountain, B.W., Feng, L., Omelchenko, M.V., Hou, S., Saito, J.A., Stott, M.B., Li,
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D., Zhao, G., Wu, J., Galperin, M.Y., Koonin, E.V., Makarova, K.S., Wolf, Y.I., Rigden,
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D.J., Dunfield, P.F., Wang, L. & Alam, M. (2008). Encapsulated in silica: genome,
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proteome and physiology of thermophic bacterium A. flavithermus WK1. Genome Biology, 9 (11), R161.
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Scott, S. A., Brooks, J. D., Rakonjac, J., Walker, K. M. R., & Flint, S. H. (2007). The
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formation of thermophilic spores during the manufacture of whole milk powder.
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International Journal of Dairy Technology, 60 (2), 109-117.
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Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994). CLUSTAL W: improving the
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sensitivity of progressive multiple sequence alignment through sequence weighting, 18
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position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22,
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4673-4680.
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phylogenetic study. Journal of Bacteriology, 173 (2), 697–703.
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Weisburg WG, Barns SM, Pelletier DA et al. (1991). 16S ribosomal DNA amplification for
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Table 1 Strains of the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) collection used in this study and evaluation of species-specificity of the PCR primers. Species-specific PCR with primer pair: Anx-RpoB5f/ Anx-RpoB1bisr
+ + + + + -
+ + -
RI PT
15939T 17956T 15730T 15866T 23212T 2641T 21510 23293 14988T 19169T 12423T 17127T 22626T 23211T 16325T 17141T 17075T 7T 31 T 1T 12T 13T 27T 10599T 10T 14992T 13864T 795T 4928T 12041T 16016T 18751T 7263T 22T 297 456 1550 2027 13552T 16325T 730T 465T 2542T 5366T 14590T 23175T 521T 1974T 15220T 24T 14349T 16487T 7060T
Fits2/Rits2
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Anoxybacillus amylolyticus Anoxybacillus bogrovensis Anoxybacillus caldiproteoliticus Anoxybacillus contaminans Anoxybacillus eryuanensis Anoxybacillus flavithermus subsp. flavithermus Anoxybacillus flavithermus subsp. flavithermus Anoxybacillus flavithermus subsp. yunnanensis Anoxybacillus kamchatkensis Anoxybacillus mongoliensis Anoxybacillus pushchinoensis Anoxybacillus rupiensis Anoxybacillus salavatliensis Anoxybacillus tengchongensis Anoxybacillus tepidamans Anoxybacillus thermarum Anoxybacillus voinovskiensis Bacillus amyloliquefaciens subsp. amyloliquefaciens Bacillus cereus Bacillus coagulans Bacillus firmus Bacillus licheniformis Bacillus pumilus Bacillus sporothermodurans Bacillus subtilis subsp. subtilis Clostridium novyi Clostridium saccharobutylicum Clostridium sporogenes Clostridium thermobutyricum Geobacillus caldoxylosilyticus Geobacillus debilis Geobacillus galactosidasius Geobacillus kaustophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus stearothermophilus Geobacillus subterraneus Geobacillus tepidamans Geobacillus thermocatenulatus Geobacillus thermodenitrificans Geobacillus thermoglucosidasicus Geobacillus thermoleovorans Geobacillus toebii Geobacillus uzenensis Moorella thermoacetica Moorella thermoautotrophica Paenibacillus graminis Paenibacillus macerans Paenibacillus turicensis Thermoanaerobacterium aciditolerans Thermoanaerobacterium saccharolyticum
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
DSM strain
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Species
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Table 2 Origin of the IF milk powder samples and molecular identification of wild strains and bulk cells analyzed in this study.
C
Trinidad and Tobago
D
Trinidad and Tobago
E
Trinidad and Tobago
F
China
G
Pakistan
H
Pakistan
+ + + + + + + + + + + + + + + + + + + + + + + + + +
RI PT
Trinidad and Tobago
Fits2/Rits2
Anx-RpoB5f/ Anx-RpoB1bisr + + + + + + + + + + + + + +
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B
Wild strain A1 Wild strain A2 Bulk A Wild strain B1 Wild strain B2 Bulk B Wild strain C1 Wild strain C2 Wild strain C3 Wild strain C4 Bulk C Wild strain D1 Wild strain D2 Wild strain D3 Wild strain D4 Wild strain E1 Wild strain E2 Wild strain E3 Bulk E Wild strain F1 Wild strain F2 Wild strain F3 Wild strain F4 Wild strain F5 Bulk F Wild strain G1 Wild strain G2 Wild strain G3 Wild strain G4 Wild strain G5 Bulk G Wild strain H1 Wild strain H2 Wild strain H3 Wild strain H4 Wild strain H5 Bulk H
Multiplex-PCR assay
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Species-specific PCR with primer pair: Anx-RpoB5f/ Fits2/Rits2 Anx-RpoB1bisr + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
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DNA from:
EP
Origin
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16S rRNA sequencing
Accession n°
G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 100% G. stearothermophilus 100% G. stearothermophilus 100% G. stearothermophilus 100% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% G. stearothermophilus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% G. stearothermophilus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% -
NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR026516 NR026516 NR040794 NR026516 NR026516 NR040794 NR040794 NR026516 NR026516 NR026516 NR026516 NR040794 NR026516 NR026516 NR026516 -
ACCEPTED MANUSCRIPT
L
Germany
M
N
O
P
Dominica Republic
Vietnam
Philippines
Morocco
RI PT
Trinidad and Tobago
-
SC
K
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
M AN U
Indonesia
-
TE D
J
EP
Trinidad and Tobago
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
AC C
I
Wild strain I1 Wild strain I2 Wild strain I3 Wild strain I4 Wild strain I5 Bulk I Wild strain J1 Wild strain J2 Bulk J Wild strain K1 Wild strain K2 Bulk K Wild strain L1 Wild strain L2 Wild strain L3 Wild strain L4 Wild strain L5 Bulk L Wild strain M1 Wild strain M2 Wild strain M3 Wild strain M4 Wild strain M5 Wild strain M6 Bulk M Wild strain N1 Wild strain N2 Wild strain N3 Wild strain N4 Wild strain N5 Wild strain N6 Bulk N Wild strain O1 Wild strain O2 Wild strain O3 Wild strain O4 Wild strain O5 Bulk O Wild strain P1 Wild strain P2 Wild strain P3 Wild strain P4 Wild strain P5
G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% G. stearothermophilus 99% Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Not sequenced Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Aeribacillus pallidus 97% Not sequenced Not sequenced Bacillus smithii 99% Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Not sequenced Not sequenced
NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 NR040794 FN428649
FN428653
KC119567
GQ293457 FN428649
FN428694
ACCEPTED MANUSCRIPT
U
V
Sri Lanka
Mexico
Indonesia
W
Indonesia
X
Pakistan
Not sequenced Not sequenced Bacillus smithii 99% Not sequenced Not sequenced Brevibacillus thermoruber 99% Not sequenced Not sequenced Not sequenced Not sequenced Bacillus smithii 99% Not sequenced G. stearothermophilus 99% Not sequenced Not sequenced Not sequenced G. stearothermophilus 100% Not sequenced Not sequenced Bacillus coagulans 99% A. flavithermus 99% G. stearothermophilus 100% Not sequenced Not sequenced Bacillus smithii 99% Aeribacillus pallidus 97% Not sequenced Not sequenced Not sequenced A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% A. flavithermus 99% -
RI PT
+ + + + + + + +
SC
+ + + + + + + -
M AN U
T
Pakistan
TE D
S
Brazil
+ + + + + + + +
EP
R
Spain
+ + + + + + + -
AC C
Q
Bulk P Wild strain Q1 Wild strain Q2 Wild strain Q3 Wild strain Q4 Wild strain Q5 Bulk Q Wild strain R1 Wild strain R2 Wild strain R3 Wild strain R4 Wild strain R5 Bulk R Wild strain S1 Wild strain S2 Wild strain S3 Wild strain S4 Wild strain S5 Bulk S Wild strain T1 Wild strain T2 Wild strain T3 Wild strain T4 Wild strain T5 Bulk T Wild strain U1 Wild strain U2 Wild strain U3 Wild strain U4 Wild strain U5 Bulk U Wild strain V1 Wild strain V2 Wild strain V3 Wild strain V4 Bulk V Wild strain W1 Wild strain W2 Wild strain W3 Bulk W Wild strain X1 Bulk X
-
EU652724
AY196006
EU652724 FN428694
FN428653
AB682458 KC429571 FN428694
EU652724 KC119567
KC429571 KC429571 KC429571 KC429571 -
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…ACGC …ACGC …ATGC …ACGT …ACGT …ACGT …GCGT …ACGT …ACGT …ACGT …ACGT …ATGT …ATGT …ACGT …ACGT …ACGT …ACGT
167 188 GCGACTTGCAGAAGACGGCACA GCGACTTGCAGAAGACGGCACA CAGAATTGCTGAGGATGGAACA GCCAGTAGCAGAGGATGGCACG TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGCAACA TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGGAACA TCCGATTGCGGAAGATGGAACA TCCAATTACGGAAGATGGAACG TCCGATTGCCGAAGATGGAACG TCCGATTGCCGAAGATGGAACG TCCGATTGCGGAAGATGGAACG TCCGATTGCGGAAGATGGGACG TCCGATTGCGGAAGATGGAACA ACCGCTTGCGGAGGACGGCACG
1 A. amylolyticus DSM15939 (JF279467.1) 2 A. contaminans DSM15866 (JF279478.1) 3 A. rupiensis DSM17127 (JF279471.1) 4 A. voinovskiensis NCIMB13956 (JF279483.1) 5 A. kamchatkensis DSM14988 (JF279479.1) 6 Anoxybacillus spp. DSM21706 (JQ397277.1) 7 A. thermarum DSM17141 (JF279472.1) 8 A. ayderensis NCIMB13972 (JF279476.1) 9 A. gonensis NCIMB13933 (JF279473.1) 10 A. salavatliensis DSM22626 (JQ397278.1) 11 A. pushchinoensis DSM12423 (JF279477.1) 12 A. eryuanensis KCTC13720 (JF279469.1) 13 A. mongoliensis DSM19169 (JQ397279.1) 14 A. tengchongensis KCTC13721 (JF279470.1) 15 A. flavithermus DSM2641 (JF279475.1) 16 A. kestanbolensis NCIMB13971 (JF279474.1) 17 A. bogrovensis DSM17956 (JF279468.1)
…TCGT …TCGT …TCGT …TTGT …TTGT …TTGT …TTGT …TTGT …TTGT …ATGT …TTGT …TCGT …TCGT …TCGT …TTGT …TCGT …TCGT
286 308 CTCTGCAGCGACAGCGTGTATC TTCTGCAGCGACAGCGTGTATC ATCAGCGGCGACTGCGTGTATT ATCAGCAGCAACAGCTTGTATC GTCGGTGGCGACAGCGTGCATT GTCGGTGGCGACAGCGTGCATT ATCGGTGGCGACAGCGTGCATT GTCGGTGGCGACAGCGTGCATT GTCGGTAGCGACAGCGTGCATT GACATTAGCGACAGATCGCATT GTCAGTTGCGACTGCATGCATT GTCCGTAGCGACGGCTTGCATT GTCCGTAGCGACGGCTTGCATT GTCCGTAGCGACAGCCTGCATT ATCGGTAGCCACAGCGTGTATC ATCCGTAGCTACAGCGTGTATC CTCGGCAGCGACAGCGTGCATC
TT… TT… TT… TT… TT… TT… TT… TT… TT…
M AN U
SC
RI PT
1 A. amylolyticus DSM15939 (JF279467.1) 2 A. contaminans DSM15866 (JF279478.1) 3 A. rupiensis DSM17127 (JF279471.1) 4 A. voinovskiensis NCIMB13956 (JF279483.1) 5 A. kamchatkensis DSM14988 (JF279479.1) 6 Anoxybacillus spp. DSM21706 (JQ397277.1) 7 A. thermarum DSM17141 (JF279472.1) 8 A. ayderensis NCIMB13972 (JF279476.1) 9 A. gonensis NCIMB13933 (JF279473.1) 10 A. salavatliensis DSM22626 (JQ397278.1) 11 A. pushchinoensis DSM12423 (JF279477.1) 12 A. eryuanensis KCTC13720 (JF279469.1) 13 A. mongoliensis DSM19169 (JQ397279.1) 14 A. tengchongensis KCTC13721 (JF279470.1) 15 A. flavithermus DSM2641 (JF279475.1) 16 A. kestanbolensis NCIMB13971 (JF279474.1) 17 A. bogrovensis DSM17956 (JF279468.1)
AT… TT… TT… TT… TT… TT… TT… TT…
CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC… CC…
AC C
EP
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Figure 1 - Sequence alignment of portions of rpoB gene sequences of A. flavithermus and other Anoxybacillus species. The accession numbers of the sequences used for the alignment are indicated. Variable sequences are highlighted in white. The sequences used for primer design are boxed.
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Figure 2 – Species-specific PCR assay by using the primer pair: Fits2/Rits2. Panel A: species-specific PCR assay by applying Prevost et al. (2010) PCR conditions (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, G. stearothermophilus DSM22T; lane 2, G. subterraneus DSM13552T; lane 3, G. caldoxylosilyticus DSM12041T; lane 4, G. thermodenitrificans DSM465T; lane 5, G. kaustophilus DSM7263T; lane 6, Clostridium thermobutyricum DSM4928T; lane 7, A. flavithermus DSM2641T; lane 8, Bacillus subtilis DSM10T; lane 9, negative control (deionized water); M, 1Kb Plus DNA Ladder. Panel B: species-specific PCR assay by applying PCR conditions developed in this study (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, G. stearothermophilus DSM22T; lane 2, G. subterraneus DSM13552T; lane 3, G. uzenensis DSM23175T; lane 4, G. caldoxylosilyticus DSM12041T; lane 5, G. thermoleovorans DSM5366T; lane 6, G. thermodenitrificans DSM465T; lane 7, G. kaustophilus DSM7263T; lane 8, Clostridium thermobutyricum DSM4928T; lane 9, Thermoanaerobacterium aciditolerans DSM16487T; lane 10, A. flavithermus DSM2641T; lane 11, Bacillus cereus DSM31T; lane 12, Bacillus subtilis DSM10T; M, 1Kb Plus DNA Ladder.
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Figure 3 – Sensitivity of the PCR assays. Upper part: Species-specific PCR assay with G. stearothermophilus DSM22T DNA as template; from left to the right: M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, 10 ng of DNA; lane 2, 5 ng of DNA; lane 3, 500 pg of DNA; lane 4, 50 pg of DNA; lane 5, 5 pg of DNA; lane 6, 0.5 pg of DNA; lane 7, 50 fg of DNA; lane 8, negative control; M, 1Kb Plus DNA Ladder (Invitrogen). Lower part: Species-specific PCR assay with A. flavithermus DSM2641T DNA as template (in duplicate); from left to the right: M, 1Kb Plus DNA Ladder (Invitrogen); lane 1-2, 10 ng of DNA; lane 3-4, 5 ng of DNA; lane 5-6, 500 pg of DNA; lane 7-8, 50 pg of DNA; lane 9-10, 5 pg of DNA; lane 11-12, 0.5 pg of DNA; M, 1Kb Plus DNA Ladder (Invitrogen).
ACCEPTED MANUSCRIPT M
1
2
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10 11 12 13 14 M
bp
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650
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Panel A
M
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8
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650 300 200 100
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M
650
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Panel C
Figure 4 – Multiplex-PCR profiles obtained with bulk cells collected from 23 milk powder samples. Panel A (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, bulk cell A; lane 2, bulk cell B; lane 3, bulk cell C; lane 4, bulk cell E; lane 5, bulk cell F; lane 6, bulk cell G; lane 7, bulk cell H; lane 8, bulk cell I; lane 9, bulk cell J; lane 10, bulk cell K; lane 11, bulk cell L; lane 12, G. stearothermophilus DSM22T; lane 13, A. flavithermus DSM2641T; lane 14, negative control; M, 1Kb Plus DNA Ladder (Invitrogen). Panel B (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, bulk cell M; lane 2, bulk cell N; lane 3, bulk cell O; lane 4, bulk cell P; lane 5, bulk cell Q; lane 6, G. stearothermophilus DSM22T; lane 7, A. flavithermus DSM2641T; lane 8, negative control. Panel C (from left to the right): M, 1Kb Plus DNA Ladder (Invitrogen); lane 1, bulk cell R; lane 2, bulk cell
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S; lane 3, bulk cell T; lane 4, bulk cell U; lane 5, bulk cell V; lane 6, bulk cell W; lane 7, bulk cell X; lane 8, G. stearothermophilus DSM22T; lane 9, A. flavithermus DSM2641T; lane 10, negative control.
ACCEPTED MANUSCRIPT
HIGHLIGHTS G. stearothermophilus and A. flavithermus identifications were achieved by PCR assays
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Primers targeting ITS 16S-23S rRNA region and rpoB gene sequence were employed
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After testing their specificity, they were combined in a Multiplex-PCR assay
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The simultaneous detection of the two target species was successfully achieved
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Rapidity (~ 4h from DNA isolation to results) of the assays emphasize their advantage
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