Diagnostic Microbiology and Infectious Disease 79 (2014) 135–140
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A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens☆ Mark S. Rundell a, Maneesh Pingle a, Sanchita Das b, Aashiq Hussain a, Oksana Ocheretina b, e, Macarthur Charles b, e, Davise H. Larone c, Eric D. Spitzer d, Linnie Golightly b, Francis Barany a,⁎ a
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Box 62, New York, NY 10021 Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, Box 62, New York, NY 10021 c Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, Box 62, New York, NY 10021 d Department of Pathology, Stony Brook University Medical Center, Stony Brook, NY 11794 e Groupe Haitien d'Étude du Sarcome de Kaposi et des Infections Opportunistes, Port-Au-Prince, Haiti b
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 17 February 2014 Accepted 26 February 2014 Available online 12 March 2014 Keywords: Molecular diagnostics Multiplex Infectious disease Enteric pathogens
a b s t r a c t Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Enteric pathogens that cause gastroenteritis remain a major global health concern and are 1 of the top infectious causes of morbidity and mortality worldwide (Cunningham et al. 2010; de Boer et al. 2010; Gomez-Duarte et al. 2009; Nguyen et al. 2005; O'Leary et al. 2009). Infectious gastroenteritis is responsible for 1.7–2.5 million deaths annually, mostly of children under 5 years of age in developing countries (Gomez-Duarte et al. 2009; Kosek et al. 2003, 2009; Liu et al. 2011; UN 2005; WHO 2011). In light of major global infectious disease emergencies, such as the cholera epidemic in Haiti and the highly
☆ Funding: This work was supported by Public Service grants UCI-AI062579 and U01AI075470-01 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. This work was also supported by Ruth L. Kirschstein National Service Award (NRSA) T32 Training Grant 5T32A1007613 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. ⁎ Corresponding author. Tel.: +1-212-746 6509; fax: +1-212-746-8104. E-mail address: [email protected] (F. Barany). http://dx.doi.org/10.1016/j.diagmicrobio.2014.02.022 0732-8893/© 2014 Elsevier Inc. All rights reserved.
virulent Escherichia coli outbreak across Europe, enteric bacterial pathogens remain a significant public health threat in both the developed and developing parts of the world (Askar et al., 2011; Enserink 2011; Walton and Ivers 2011; Nature News, 2011). Conventional diagnostic procedures for routine detection of enteric bacterial pathogens entail enrichment steps, selective culture, biochemical identification, serotyping, and/or resistance profiling. The steps for pathogen identification are laborious and time consuming, often taking 3–5 days for a final result. Several pathogens are indistinguishable from normal flora by morphology and biochemical properties. Other pathogens are difficult to culture while some have limited viability outside the host (Cunningham et al. 2010; de Boer et al. 2010; Schuurman et al. 2007). Molecular methods, including TaqMan assays or antigen detection tests, offer more rapid and sensitive detection than conventional culture methods (Gomez-Duarte et al. 2009; Liu et al. 2011; O'Leary et al. 2009). However, these molecular methods are limited in scope and scale for both the number of samples processed annually and the broad and wide-ranging number of possible pathogens that can be detected.
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We have developed, characterized, and applied a high-throughput multiplex molecular assay for the simultaneous detection of Campylobacter spp. (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter fetus, Campylobacter upsaliensis), Vibrio spp. (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio furnissii, Vibrio metschnikovii, Vibrio hollisae), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC), Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella boydii), Salmonella spp. (Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Salmonella arizoniae, Salmonella cholerasuis), Listeria monocytogenes, and Yersinia enterocolitica. This assay can detect and identify over 29 species of enteropathogenic bacteria across 7 different genera in a single assay protocol. Additionally, we applied this assay to clinically significant culture-positive samples for V. cholerae obtained from our international study site, the Groupe Haitien d’Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port-au-Prince, Haiti. Currently, this multiplex assay is the only comprehensive molecular test reported to date for the detection of all category B bacterial food and water-borne pathogens.
2.3. Preparation of seeded clinical specimens Approximately 1000 individual stool specimens that were submitted as part of diagnostic evaluations and were negative by routine culture at NYPH/WCMC were used for seeding with individual clinical isolates of the category B bacterial pathogens. Stool specimens were stored in a cold room at 4 °C. Pure cultures of clinical isolates were inoculated into 1 mL of trypticase soy broth. Ten-fold serial dilutions of the bacterial suspensions were prepared in trypticase soy broth, and the CFU/mL was estimated by counting the number of colonies on blood agar plates streaked with 10 μL aliquots of each dilution and incubated overnight. 100 μL of the individual bacterial suspensions at known concentrations was spiked into 200 mg of culture-negative stool specimens for determination of the analytical sensitivity. The concentration range tested was 1 × 10 1 to 1 × 10 9 CFU/mL. The concentration at which all pathogens were detected was selected for subsequent seeding of culture-negative clinical specimens to determine the diagnostic sensitivity and specificity of the PCR/LDR assay.
2.4. DNA extraction 2. Materials and methods 2.1. Bacterial isolates and clinical specimens Clinical isolates from stool and other clinical specimens that had been previously phenotypically identified using conventional microbiological techniques were obtained from the clinical microbiology laboratory at New York-Presbyterian Hospital/Weill Cornell Medical Center (NYPH/WCMC) and the New York City Department of Health. Molecularly characterized isolates of diarrheagenic E. coli were kindly provided by Dr Lee Riley of the University of California Berkeley. Pure cultures of the following strains were obtained from the American Type Culture Collection (ATCC): V. mimicus (ATCC 33655), V. metschnikovii (ATCC 7708), V. fluvialis (ATCC 33809), V. parahaemolyticus (ATCC BAA-241), V. furnissii (ATCC 35627), E. coli serotype O124 (ATCC 43893), E. coli serotype O111 (ATCC 43887), E. coli serotype O78:H11 (ATCC 35401), S. boydii (ATCC 25930), C. lari (ATCC BAA-1060), and C. coli (ATCC 43485). A total of 1042 culture-negative stool specimens from hospital patients were obtained from the clinical microbiology laboratory at NYPH/WCMC between October 2008 and May 2010. These specimens were subject to routine screening for the presence of C. jejuni, Salmonella spp., and Shigella spp., as well as any additional organisms requested by a physician. Four stool specimens culture-positive for Salmonella spp. (n = 1), C. jejuni (n = 2), and STEC (n = 1) were collected at NYPH/WCMC during the same time period. Forty additional specimens (25 stools and 15 rectal swabs) that were culture-positive for V. cholerae were obtained from GHESKIO. These samples were collected in November 2010 from patients with suspected cholera infection during the V. cholerae epidemic in Haiti. Swabs were transported in 0.5 mL 0.9% sterile saline. All samples were held at 4 °C until processed for DNA extractions. Human experimentation guidelines of the United States Department of Health and Human Services and those of NYPH/WCMC were followed in the conduct of clinical research. 2.2. Preparation of clinical isolates A total of 97 clinical isolates were grown overnight at 35 °C on sheep blood agar, and growth was checked for purity. Three to 5 colonies of each isolate were suspended in 100 μL of phosphate buffered saline (PBS) buffer pH 7.5. Suspensions of each clinical isolate were prepared in quadruplicate in PBS and transferred to a 96-well deep well plate for extraction of DNA.
Total genomic DNA was extracted from pure cultures and fecal specimens using the QIAamp DNA Stool Mini kit (Qiagen, Inc., Valencia, CA, USA), with minor modifications. To accommodate the high sample numbers to be analyzed and the high-throughput design of our downstream molecular assays, we incorporated QIAamp 96Well Plates from the QIAamp 96 DNA Blood Kits into the protocol, replacing the individual QIAamp Mini Spin Columns. All specimens were processed according to the manufacturer’s instructions in a 96well format.
Table 1 List of genetic targets used to identify each pathogen. Pathogen
Genes of interest
LDR product detection schemea
EPEC EAEC DAEC ETEC
bfp aggR daa eltB (LT) estA (ST) stx1 virA virF invA spvC clyA rpo rpo rpo rpo rpo rpo rpo 16s rDNA hipO 16s rDNA asp 16s rDNA 16s rDNA 16s rDNA invA ail 16s rDNA
Zip4-Cy3; Zip5-Cy3; Zip6-Cy3 Zip1-Cy3; Zip2-Cy3; Zip3-Cy3 Zip13Cy3; Zip14-Cye3; Zip15-Cy3 Zip19-Cy3; Zip20-Cy3; Zip21-Cy3 Zip22-Cy3; Zip23-Cy3; Zip13-Cy5 Zip34-Cy3; Zip35-Cy3; Zip36-Cy3 Zip18-Cy5; Zip19-Cy5; Zip20-Cy5 Zip21-Cy5; Zip22-Cy5; Zip23-Cy5 Zip10-Cy3; Zip11-Cy3; Zip12-Cy3 Zip16-Cy3; Zip17-Cy3; Zip18-Cy3 Zip15-Cy5; Zip16-Cy5; Zip17-Cy5 Zip1-Cy5; Zip5-Cy5; Zip9-Cy5 Zip1-Cy5; Zip6-Cy5; Zip9-Cy5 Zip1-Cy5; Zip4-Cy5; Zip12-Cy5 Zip1-Cy5; Zip6-Cy5; Zip12-Cy5 Zip2-Cy5; Zip8-Cy5; Zip10-Cy5 Zip3-Cy5; Zip5-Cy5; Zip11-Cy5 Zip7-Cy5; Zip10-Cy5; Zip14-Cy5 Zip24-Cy5; Zip25-Cy5 Zip38-Cy5; Zip39-Cy5; Zip41-Cy5 Zip24-Cy5; Zip25-Cy5 Zip42-Cy5; Zip43-Cy5; Zip44-Cy5 Zip24-Cy5; Zip25-Cy5; Zip33-Cy5 Zip34-Cy5; Zip35-Cy5; Zip36-Cy5 Zip29Cy5; Zip30-Cy5; Zip31-Cy5 Zip45-Cy5; Zip46-Cy5; Zip47-Cy5 Zip37-Cy5; Zip40-Cy5; Zip48-Cy5 Zip27-Cy3; Zip28-Cy3; Zip32-Cy3
STEC EIEC, Shigella spp.b Salmonella spp. S. typhi V. cholerae V. mimicus V. furnissii V. fluvialis V. metschnikovii V. parahaemolyticus V. hollisae C. jejuni C. coli C. lari C. upsaliensis C. fetus Y. enterocolitica L. monocytogenes a
LDR products labeled with VeraCode sequence number (Zip) on the 5′-end and the fluorescent dye (Cy3 or-Cy5) labeled on the 3′-end. Up to 3 species-specific LDR products for each genetic target. b EIEC and Shigella spp. share the same genetic targets for identification and are not distinguished from each other.
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2.5. Primer design PCR and LDR primers were designed using Oligo 6.0™ software (Molecular Biology Insights, Cascade, CO, USA). PCR primers were designed based on sequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov) as previously described (Pingle et al. 2007). A list of the genetic targets chosen for primer design is shown in Table 1. A total of 51 PCR primers were designed for all category B bacterial pathogens (Supplemental Table 1). All PCR primers were designed with similar melting temperatures (Tm) to amplify each target gene to ensure accurate identification despite organism-specific gene variations. PCR primers were optimized and validated in uniplex and multiplex reactions for each genetic target. To account for genetic variability within a target genetic sequence, in some instances, multiple PCR primer pairs were generated to amplify a single amplicon. The specificity of the PCR primers in selectively amplifying the desired pathogens was tested in silico by BLAST analysis of the primer sequences against the bacterial sequence databases. Each PCR amplicon was detected by a subsequent LDR reaction. LDR is a linear amplification technique, where 2 adjacent oligonucleotides hybridize
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to a single DNA strand and are ligated by a high fidelity thermostable ligase when there is a perfect match at the junction point. LDR primers were designed to distinguish genus-specific and species-specific single base variants within each PCR amplicon. For example, the Zip25-Cy5 and Zip 24-Cy5 signals are generated from single nucleotide polymorphisms (SNP) that are common to C. jejuni, C. coli, and C. lari (see Table 1). These can be distinguished from each other on the basis of the unique signature generated from the combination of signals (e.g., the unique signature for C. jejuni is “Zip24-Cy5; Zip25-Cy5; Zip38Cy5; Zip39-Cy5; Zip41-Cy5”, whereas that for C. coli is “Zip24-Cy5; Zip25-Cy5; Zip42-Cy5; Zip43-Cy5; Zip44-Cy5”). The upstream LDR primers had melting temperatures of 70 °C and were labeled at the 5′end with a unique VeraCode sequence and C12 amino blocking group. The downstream LDR primers were phosphorylated at the 5′-end, fluorescently labeled at the 3′-end with either a Cy3 dye or Cy5 dye, and had Tm values in the range of 70–75 °C. The 157 LDR primers were optimally partitioned into 2 LDR reactions to prevent primer dimer interactions and ligation deficiency and are shown in Supplemental Table 2. Up to 3 LDR products were designed to detect species-specific single base variants for each genetic target (Table 1). Both PCR and LDR
Fig. 1. Schematic of the PCR/LDR assay with molecular readout using the BeadXpress and Veracode platform. Gene-specific PCR primer pairs were designed to amplify their distinct genetic targets if present. Each PCR amplicon ranged in size from 400 to 600 base pairs. Within each PCR amplicon, we then designed LDR primer pairs to identify single base variants at multiple locations to allow us to distinguish between different pathogenic bacteria. At any given single base variant, the allele specific upstream LDR primers are designed to ligate to the downstream primer only if there is a perfect match at the junction point. The upstream LDR primers bear zipcode complement sequences and amino blocking groups on the 5′end, while the downstream LDR primers have either a Cy3 or Cy5 fluorescent label. Ligation of the LDR primers results in fluorescently labeled products that are subsequently hybridized to zipcode addresses attached to veracode beads. Each bead contains a unique barcode that can be scanned by the BeadXpress reader. Detection of the bead and fluorescent signal scores for the presence of the microbial pathogen.
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primers were synthesized with degenerate bases where required due to sequence variation. PCR and LDR primers were obtained from Integrated DNA Technologies (Coralville, IA, USA).
Hybridization was carried out at 45 °C for 1 h with agitation (1000 rpm). VeraCode Bead Plates were scanned by the BeadXpress Reader System.
2.6. PCR/LDR assay
2.7. Data analysis
A schematic of the PCR/LDR assay is shown in Fig. 1. PCR amplifications were carried out in a total volume of 25 μL containing GeneAmp 1X PCR Buffer II (Applied Biosystems, Foster City, CA, USA), 2.5 mmol/L MgCl2, 200 μmol/L of each dNTP, 5 pmol of each PCR primer, 0.5 ng/nL BSA (Sigma, Saint Louis, MO, USA), 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), and 2 μL of template DNA. Samples were thermocycled using the following parameters: 10 min at 95 °C, followed by 35 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min), and a final extension at 72 °C for 7 min followed by 99.9 °C for 30 min to destroy the polymerase before being held indefinitely at 4 °C. Two separate LDR reactions were prepared containing 500 fmol/μL of each of the appropriate upstream and downstream LDR primers. An aliquot of each primer mix was separately kinased prior to its use in LDR reactions as previously described (Pingle et al. 2007; Rodas et al. 2009; Rondini et al. 2008). LDR reactions were carried out as previously described (Pingle et al. 2007; Rodas et al. 2009; Rondini et al. 2008). Following the LDR reactions, the products were treated with 3′-5′ and 5′-3′ exonucleases to destroy unligated LDR primers. Exonuclease reactions were performed in a total volume of 30 μL containing 1 × 10 4 units of exonuclease I, 2.5 × 10 3 units of Lambda Exonuclease, 1X exonuclease I buffer, and 20 μL of LDR products. Samples were thermocycled using the following parameters: 37 °C for 60 min followed by 80 °C for 10 min before being held indefinitely at 4 °C. VeraCode microbeads (Illumina, San Diego, CA, USA) were prepared according to the manufacturer’s instructions. A total of 48 VeraCode microbeads were mixed and distributed into 96-well microplates using the VeraCode Bead Kitting System in 70% ethanol. Following aspiration of ethanol, 150 μL of 1× saline sodium citrate (SSC) and 0.05% Tween 20 was added to each well. Approximately 100 μL of the buffer was aspirated using the 8-pin aspirator manifold. A total of 30 μL of the LDR products was added to each well.
Signal intensity data obtained from the BeadXpress Reader System were analyzed on Excel spreadsheets. The data were normalized using the median signal values of the negative controls. Signal intensities ≥3-fold higher than the background were considered positive. All samples were tested by the PCR/ LDR assay in quadruplicate. An LDR reaction was considered to be positive when 2 or more of the replicates were positive. Positivity for at least 2 different LDR targets was required for an organism identification. 2.8. Resolution of discordant results Specimens that were PCR/LDR-negative but culture-positive or seeded with a known organism were subjected to repeat DNA extraction and repeat PCR/LDR assay. PCR products were also analyzed by 2% agarose gel electrophoresis to determine if falsenegative PCR/LDR results were due to failure of the PCR step or the LDR step. Culture-positive specimens in which PCR/LDR detected an additional organism were subjected to repeat DNA extraction, repeat PCR/LDR assay, and DNA sequencing. Seeded specimens that were PCR/LDR-positive for an organism that had not been seeded were subjected to repeat DNA extraction, repeat PCR/LDR assay, and DNA sequencing from their corresponding unseeded original specimens. 2.9. Sequencing of discordant samples DNA extracted from discordant samples was subjected to sequencing using the same PCR primers designed for the PCR/LDR assay. PCR reactions were carried out as described above except that a single amplicon was generated in each PCR by using only 1 pair of amplification primers. The PCR products were purified using QIAQuick PCR purification plates (Qiagen) according to the
Table 2 Diagnostic sensitivity and specificity of the PCR/LDR assay on seeded clinical specimens. Organism (no. of seeded specimens/no. of strains) C. jejuni (36/2) Shigella spp. (85/11) Salmonella spp. (165/24) S. typhi (90/7) Y. enterocolitica (29/6) L. monocytogenes (214/22) V. fluvialis (11/1) V. parahaemolyticus (21/1) V. furnissii (21/1) V. mimicus (18/1) V. metschnikovii (22/1) C. coli (63/2) C. lari (21/2) V. cholerae (4/1) EIEC (30/2) STEC (62/7) ETEC (33/2) DAEC (21/1) EPEC (12/3) a b c d
Specimens seeded with listed organism PCR/LDR-posa
PCR/LDR-nega
36 83 163 88 29 206 11 21 21 18 22 63 19 4 30 60 33 21 12
0 2 2 2 0 8 0 0 0 0 0 0 2 0 0 2 0 0 0
Sensitivity (%)
100 97.6 98.8 97.8 100 96.3 100 100 100 100 100 100 90.5 100 100 96.8 100 100 100
Specimens not seeded with listed organismb PCR/LDR-posa
PCR/LDR-nega
0 0 0 0 8 (2)c 1 0 4 (1)c 4 0 0 3 (1)c 1 (1)c 0 4 6 (1)c 1 (1)c 16 (10)c 4
1006 957 877 952 1006 835 1031 1018 1017 1024 1020 978 1020 1038 1008 976 1008 1005 1026
Specificity (%)
100 100 100 100 99.4d 99.9 100 99.7d 99.6 100 100 99.7d 100d 100 99.6 99.5d 100d 99.4d 99.6
PCR/LDR positive or negative for the specified organism. Includes stool specimens that were seeded with another organism and 84 stool specimens that were not seeded. Number in parentheses indicates the number of specimens in which the same organism was detected in the underlying unspiked specimen by PCR and sequencing. Specificity was calculated based on the number of unconfirmed positives.
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manufacturer’s instructions. PCR products were inspected for purity by electrophoresis on 2% agarose gels as well as by measuring the absorbance at 260 and 280 nm. The purified PCR products were adjusted to concentrations of 3–5 ng/μL and sent to Macrogen, Inc. (Rockville, MD, USA) for sequencing. The sequence data obtained were queried against the GenBank database using BLAST (http://www.ncbi.nlm.nih. gov/BLAST). The highest scoring matches were recorded for each organism. 3. Results 3.1. Analytical sensitivity and specificity of the PCR/LDR assay with clinical isolates The analytical specificity of the assay was 100% for pure cultures of all 97 clinical isolates. There were no false positives when testing each pure culture isolate with the PCR/LDR assay, indicating no crossreaction between the 51 PCR primers and 157 LDR primers. We determined the limit of detection of our assay on seeded specimens to range from 10 1 CFU/mL to 10 5 CFU/mL. The concentration in which all pathogens were detected was 10 5 CFU/mL. 3.2. Diagnostic sensitivity and specificity of PCR/LDR with seeded specimens The diagnostic sensitivity and specificity of the PCR/LDR assay were assessed blindly on a large collection of 958 stool specimens that were culture-negative for C. jejuni, Salmonella spp., and Shigella spp. Specimens were individually spiked with 1 × 10 5 CFU/mL of category B agents and then tested by PCR/LDR. An additional 84 culturenegative stool specimens were tested without spiking. The results are shown in Table 2. C. jejuni, Salmonella spp., and Shigella spp. were detected with an overall sensitivity of 98.8% and a specificity of 100% (Table 2). There were 6 false-negative results (Shigella spp., n = 2; Salmonella spp., n = 2; S. typhi, n = 2). Agarose gel electrophoresis revealed an absence of PCR products in these reactions indicating that PCR inhibition was the probable cause for these false-negative results. Stool specimens are known to have high concentrations of PCR inhibitors, such as complex polysaccharides and carbohydrates, heme groups, bile acids, and bilirubins (Monteiro et al. 1997; Nechvatal et al. 2008). The PCR/LDR assay gave similar results for the remaining category B agents including other Campylobacter spp., Vibrio spp., Yersinia enterocolitica, L. monocytogenes, and pathogenic E. coli (EIEC, STEC, ETEC, DAEC, and EPEC); however, there were more discrepant results. The overall sensitivity for these organisms was 97.9% (for 12/15 species the sensitivity was 100%). The assay was unable to detect the seeded organisms in 12 specimens (L. monocytogenes, n = 8; C. lari, n = 2; and STEC, n = 2). PCR/LDR was repeated on these samples and was negative. The absence of PCR products for each false-negative result again suggested that PCR inhibition was the probable cause. In 52 of the seeded specimens, PCR/LDR detected a second organism in addition to the seeded organism. Six of these discordant results involved detection of an organism that had been seeded into an adjacent well within a 96-well microplate. These presumably represent potential cross-contamination or carryover events. The remaining 46 discordant samples were subjected to repeat DNA extraction, repeat PCR/LDR assay, and DNA sequencing from their corresponding unseeded original specimens. In 17 of these samples, the second organism was detected in the original unseeded specimen by both PCR/LDR and sequencing. These specimens contained DAEC (n = 10), Y. enterocolitica (n = 2), STEC (n = 1), ETEC (n = 1), V. parahaemolyticus (n = 1), C. lari (n = 1), and C. coli (n = 1). In the other 29 discordant samples, the second organism was not detected in the unseeded specimen. This may have been caused by crosscontamination or carryover events but may also have been a
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Table 3 Culture-positive samples analyzed by PCR/LDR. Source (type) of specimens
No. of samples
GHESKIO (stool, n = 25; rectal swab, 40 n = 15) WCMC (stool) 1 WCMC (stool) WCMC (stool)
2 1
Culture result
PCR/LDR result (no.)
V. cholerae
V. cholerae (39)a
Salmonella spp. C. jejuni STEC
Negative C. jejuni (2) STEC (1)
a In 11 of these samples, PCR/LDR detected a mixed infection [DAEC (7), EIEC/Shigella spp. (1), V. furnissii (2), and V. mimicus (1)]. The presence of mixed infections was confirmed by sequencing or species-specific PCR.
consequence of prolonged storage of the specimens at 4 °C potentially resulting in DNA degradation. Based on the above analysis, the apparent specificity of PCR/LDR ranged from 99.4% to 100% (Table 2). 3.3. Application of the PCR/LDR assay on culture-positive clinical samples A total of 44 culture-positive clinical specimens were analyzed by the PCR/LDR assay. The results are shown in Table 3. Pathogen identification by the PCR/LDR assay was concordant in 42 of the 44 positive specimens for an overall sensitivity of 95.5%. Of the 44 culture-positive specimens, 1 rectal swab specimen was negative for V. cholerae, and 1 culture-positive stool specimen for Salmonella spp. was negative by the PCR/LDR assay. Repeat PCR/LDR of these 2 samples was also negative. Enrichment of the rectal swab specimen in alkaline peptone water prior to culture on thiosulfate citrate bile salts sucrose (TCBS) agar at 35 °C for 48 h could not recover V. cholerae. The specimen positive for Salmonella spp. was subjected to culture on blood agar and MacConkey agar plates at 35 °C for 48 h; no growth was observed. In 11 of the 40 culture-positive specimens for V. cholerae, PCR/LDR detected a second pathogen (DAEC, n = 7; EIEC/ Shigella spp., n = 1; V. furnissii, n = 2; and V. mimicus, n = 1). The presence of DAEC and EIEC/Shigella spp. was confirmed by sequencing. Because the genetic target to detect all Vibrio pathogens is communal, we were unable to confirm the results via sequencing due to the potential of only the more abundant genetic target being detected. Therefore, we designed species-specific PCR probes to target single base variants within the rpo gene to distinguish the various Vibrio pathogens. The presence of additional pathogens for these 3 specimens was confirmed by species-specific PCR. 4. Discussion In this study, we describe the development and application of a multiplex PCR/LDR assay for the identification of all NIAID category B bacterial food and water-borne pathogens. The assay was designed to PCR amplify 17 genetic targets attributed to a panel of 29 pathogenic bacterial species. Species-specific single base variants within these PCR amplicons are then queried using LDR primer pairs that generate ligation products that are subsequently hybridized to their discrete individual VeraCode beads for identification (Fig. 1). The LDR products produced signals based on bead hybridization and fluorescence and the combination of signals generated a unique molecular signature for each bacterial pathogen. The PCR/LDR assay was initially optimized with DNA extracted from variants of each pathogen isolated from stool or rectal swabs. A number of the pathogens in our panel are rarely encountered. Therefore, we were limited in our ability to obtain isolates or culture-positive clinical specimens for the following organisms: C. fetus, C. upsaliensis, V. hollisae, and EAEC. However, the PCR/LDR assay includes primers to detect and identify all category B bacterial pathogens, including the aforementioned 4 pathogens. A total of 97 clinical isolates representing the remaining category B pathogens were correctly identified with no cross-reactivity.
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The analytical sensitivity of our assay was determined using seeded stool specimens. All organisms were detected at concentration of 10 5 CFU/mL, consistent with other molecular assays for gastrointestinal pathogens (Liu et al. 2013). The diagnostic sensitivity and specificity of the PCR/LDR assay were assessed with culture-negative stool specimens seeded with 1 × 10 5 CFU/mL of category B pathogens. This approach was taken because the positivity rate for pathogens in stool cultures at most academic medical centers in the United States (including NYPH/WCMC) is less than 5%. A large number of separate stool specimens were used in the seeding experiments to assess the robustness of the assay with clinical samples. The sensitivity of PCR/LDR for detecting category B bacteria pathogens ranged from 90% to 100% as determined with a large collection of seeded specimens and a smaller set of specimens from Haiti that contained V. cholerae. The majority of the false-negative reactions may have been caused by PCR inhibitors. Future versions of the assay will include PCR/LDR targets that can serve as an inhibitor control. The apparent specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. The specificity was 100% for C. jejuni, Salmonella spp., and Shigella spp., organisms for which routine stool cultures have been optimized. The putative false-positive PCR/LDR results were restricted to organisms for which selective culture methods are not routinely performed (Y. enterocolitica, L. monocytogenes, and V. parahaemolyticus) or that cannot be detected with standard stool culture techniques (EIEC, DAEC, STEC, and EPEC). Other investigators have seen similar discrepancies when multiplex molecular assays and culture methods are performed on clinical samples. Liu et al. 2013 evaluated the ability of a TaqMan Array Card (TAC) assay to detect a variety of enteric pathogens present in clinical specimens from Tanzania and Bangladesh. The specificity of TAC for detecting Salmonella, V. cholerae, Shigella, and pathogenic E. coli was 71% when compared to culture-based methods but increased to 95% when compared to a PCR-Luminex amplification assay (the corresponding sensitivity values were 99% and 98%, respectively). Due to the extensive and substantial number of enteric bacterial pathogens that can be incriminated as causative agents in stool specimens from infected individuals, the etiological significance of enteric bacteria prompts the need for an assay that can simultaneously detect and identify a large comprehensive panel of pathogens. The widespread and all-inclusive adeptness of the PCR/LDR assay can have a major clinical impact, especially in the design of diagnostic algorithms and the interpretation of epidemiological studies. The PCR/LDR assay is capable of screening a large panel of pathogens to carry out such an investigation, although additional characterization studies of the PCR/LDR assay need to be carried out on the potential interference of various pathogenic loads in mixed infections. Implementation of this molecular assay affords the capability of screening a comprehensive panel of enteric pathogens with enhanced sensitivity and speed of diagnosis, providing a sampleto-result answer in less than 8 hours with an overall diagnostic sensitivity of N98%. Additional applications of PCR/LDR with a universal array readout integrated in a microfluidic device exhibited detection of pathogenic bacteria in b40 min with minimal operator intervention, revealing the potential of PCR/LDR-based molecular analysis systems to generate results rapidly in a fully automated process (Chen et al. 2012). Combining these modular approaches will provide an earlier warning of potential public health threats, as well as a more accurate surveillance and profile of food and water-borne pathogens. This multiplex molecular assay can yield accurate, timely information, resulting in improved patient care while also providing epidemiological data for infection control and virulence/toxin surveillance. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2014.02.022.
Acknowledgments We thank Stephen Jenkins, Audrey Schuetz, and the technical staff of the clinical microbiology laboratory at NYPH/WCMC for collecting and providing clinical isolates and specimens and Bill Pape at GHESKIO for his advice and support. We thank Jianmin Huang for providing the AK16D ligase enzyme. We acknowledge Lee Riley of the University of California Berkeley and the New York City Department of Health for providing us with clinical isolates.
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens☆ Mark S. Rundell a, Maneesh Pingle a, Sanchita Das b, Aashiq Hussain a, Oksana Ocheretina b, e, Macarthur Charles b, e, Davise H. Larone c, Eric D. Spitzer d, Linnie Golightly b, Francis Barany a,⁎ a
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Box 62, New York, NY 10021 Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, Box 62, New York, NY 10021 c Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, Box 62, New York, NY 10021 d Department of Pathology, Stony Brook University Medical Center, Stony Brook, NY 11794 e Groupe Haitien d'Étude du Sarcome de Kaposi et des Infections Opportunistes, Port-Au-Prince, Haiti b
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 17 February 2014 Accepted 26 February 2014 Available online 12 March 2014 Keywords: Molecular diagnostics Multiplex Infectious disease Enteric pathogens
a b s t r a c t Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Enteric pathogens that cause gastroenteritis remain a major global health concern and are 1 of the top infectious causes of morbidity and mortality worldwide (Cunningham et al. 2010; de Boer et al. 2010; Gomez-Duarte et al. 2009; Nguyen et al. 2005; O'Leary et al. 2009). Infectious gastroenteritis is responsible for 1.7–2.5 million deaths annually, mostly of children under 5 years of age in developing countries (Gomez-Duarte et al. 2009; Kosek et al. 2003, 2009; Liu et al. 2011; UN 2005; WHO 2011). In light of major global infectious disease emergencies, such as the cholera epidemic in Haiti and the highly
☆ Funding: This work was supported by Public Service grants UCI-AI062579 and U01AI075470-01 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. This work was also supported by Ruth L. Kirschstein National Service Award (NRSA) T32 Training Grant 5T32A1007613 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. ⁎ Corresponding author. Tel.: +1-212-746 6509; fax: +1-212-746-8104. E-mail address: [email protected] (F. Barany). http://dx.doi.org/10.1016/j.diagmicrobio.2014.02.022 0732-8893/© 2014 Elsevier Inc. All rights reserved.
virulent Escherichia coli outbreak across Europe, enteric bacterial pathogens remain a significant public health threat in both the developed and developing parts of the world (Askar et al., 2011; Enserink 2011; Walton and Ivers 2011; Nature News, 2011). Conventional diagnostic procedures for routine detection of enteric bacterial pathogens entail enrichment steps, selective culture, biochemical identification, serotyping, and/or resistance profiling. The steps for pathogen identification are laborious and time consuming, often taking 3–5 days for a final result. Several pathogens are indistinguishable from normal flora by morphology and biochemical properties. Other pathogens are difficult to culture while some have limited viability outside the host (Cunningham et al. 2010; de Boer et al. 2010; Schuurman et al. 2007). Molecular methods, including TaqMan assays or antigen detection tests, offer more rapid and sensitive detection than conventional culture methods (Gomez-Duarte et al. 2009; Liu et al. 2011; O'Leary et al. 2009). However, these molecular methods are limited in scope and scale for both the number of samples processed annually and the broad and wide-ranging number of possible pathogens that can be detected.
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We have developed, characterized, and applied a high-throughput multiplex molecular assay for the simultaneous detection of Campylobacter spp. (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter fetus, Campylobacter upsaliensis), Vibrio spp. (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio furnissii, Vibrio metschnikovii, Vibrio hollisae), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC), Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella boydii), Salmonella spp. (Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Salmonella arizoniae, Salmonella cholerasuis), Listeria monocytogenes, and Yersinia enterocolitica. This assay can detect and identify over 29 species of enteropathogenic bacteria across 7 different genera in a single assay protocol. Additionally, we applied this assay to clinically significant culture-positive samples for V. cholerae obtained from our international study site, the Groupe Haitien d’Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port-au-Prince, Haiti. Currently, this multiplex assay is the only comprehensive molecular test reported to date for the detection of all category B bacterial food and water-borne pathogens.
2.3. Preparation of seeded clinical specimens Approximately 1000 individual stool specimens that were submitted as part of diagnostic evaluations and were negative by routine culture at NYPH/WCMC were used for seeding with individual clinical isolates of the category B bacterial pathogens. Stool specimens were stored in a cold room at 4 °C. Pure cultures of clinical isolates were inoculated into 1 mL of trypticase soy broth. Ten-fold serial dilutions of the bacterial suspensions were prepared in trypticase soy broth, and the CFU/mL was estimated by counting the number of colonies on blood agar plates streaked with 10 μL aliquots of each dilution and incubated overnight. 100 μL of the individual bacterial suspensions at known concentrations was spiked into 200 mg of culture-negative stool specimens for determination of the analytical sensitivity. The concentration range tested was 1 × 10 1 to 1 × 10 9 CFU/mL. The concentration at which all pathogens were detected was selected for subsequent seeding of culture-negative clinical specimens to determine the diagnostic sensitivity and specificity of the PCR/LDR assay.
2.4. DNA extraction 2. Materials and methods 2.1. Bacterial isolates and clinical specimens Clinical isolates from stool and other clinical specimens that had been previously phenotypically identified using conventional microbiological techniques were obtained from the clinical microbiology laboratory at New York-Presbyterian Hospital/Weill Cornell Medical Center (NYPH/WCMC) and the New York City Department of Health. Molecularly characterized isolates of diarrheagenic E. coli were kindly provided by Dr Lee Riley of the University of California Berkeley. Pure cultures of the following strains were obtained from the American Type Culture Collection (ATCC): V. mimicus (ATCC 33655), V. metschnikovii (ATCC 7708), V. fluvialis (ATCC 33809), V. parahaemolyticus (ATCC BAA-241), V. furnissii (ATCC 35627), E. coli serotype O124 (ATCC 43893), E. coli serotype O111 (ATCC 43887), E. coli serotype O78:H11 (ATCC 35401), S. boydii (ATCC 25930), C. lari (ATCC BAA-1060), and C. coli (ATCC 43485). A total of 1042 culture-negative stool specimens from hospital patients were obtained from the clinical microbiology laboratory at NYPH/WCMC between October 2008 and May 2010. These specimens were subject to routine screening for the presence of C. jejuni, Salmonella spp., and Shigella spp., as well as any additional organisms requested by a physician. Four stool specimens culture-positive for Salmonella spp. (n = 1), C. jejuni (n = 2), and STEC (n = 1) were collected at NYPH/WCMC during the same time period. Forty additional specimens (25 stools and 15 rectal swabs) that were culture-positive for V. cholerae were obtained from GHESKIO. These samples were collected in November 2010 from patients with suspected cholera infection during the V. cholerae epidemic in Haiti. Swabs were transported in 0.5 mL 0.9% sterile saline. All samples were held at 4 °C until processed for DNA extractions. Human experimentation guidelines of the United States Department of Health and Human Services and those of NYPH/WCMC were followed in the conduct of clinical research. 2.2. Preparation of clinical isolates A total of 97 clinical isolates were grown overnight at 35 °C on sheep blood agar, and growth was checked for purity. Three to 5 colonies of each isolate were suspended in 100 μL of phosphate buffered saline (PBS) buffer pH 7.5. Suspensions of each clinical isolate were prepared in quadruplicate in PBS and transferred to a 96-well deep well plate for extraction of DNA.
Total genomic DNA was extracted from pure cultures and fecal specimens using the QIAamp DNA Stool Mini kit (Qiagen, Inc., Valencia, CA, USA), with minor modifications. To accommodate the high sample numbers to be analyzed and the high-throughput design of our downstream molecular assays, we incorporated QIAamp 96Well Plates from the QIAamp 96 DNA Blood Kits into the protocol, replacing the individual QIAamp Mini Spin Columns. All specimens were processed according to the manufacturer’s instructions in a 96well format.
Table 1 List of genetic targets used to identify each pathogen. Pathogen
Genes of interest
LDR product detection schemea
EPEC EAEC DAEC ETEC
bfp aggR daa eltB (LT) estA (ST) stx1 virA virF invA spvC clyA rpo rpo rpo rpo rpo rpo rpo 16s rDNA hipO 16s rDNA asp 16s rDNA 16s rDNA 16s rDNA invA ail 16s rDNA
Zip4-Cy3; Zip5-Cy3; Zip6-Cy3 Zip1-Cy3; Zip2-Cy3; Zip3-Cy3 Zip13Cy3; Zip14-Cye3; Zip15-Cy3 Zip19-Cy3; Zip20-Cy3; Zip21-Cy3 Zip22-Cy3; Zip23-Cy3; Zip13-Cy5 Zip34-Cy3; Zip35-Cy3; Zip36-Cy3 Zip18-Cy5; Zip19-Cy5; Zip20-Cy5 Zip21-Cy5; Zip22-Cy5; Zip23-Cy5 Zip10-Cy3; Zip11-Cy3; Zip12-Cy3 Zip16-Cy3; Zip17-Cy3; Zip18-Cy3 Zip15-Cy5; Zip16-Cy5; Zip17-Cy5 Zip1-Cy5; Zip5-Cy5; Zip9-Cy5 Zip1-Cy5; Zip6-Cy5; Zip9-Cy5 Zip1-Cy5; Zip4-Cy5; Zip12-Cy5 Zip1-Cy5; Zip6-Cy5; Zip12-Cy5 Zip2-Cy5; Zip8-Cy5; Zip10-Cy5 Zip3-Cy5; Zip5-Cy5; Zip11-Cy5 Zip7-Cy5; Zip10-Cy5; Zip14-Cy5 Zip24-Cy5; Zip25-Cy5 Zip38-Cy5; Zip39-Cy5; Zip41-Cy5 Zip24-Cy5; Zip25-Cy5 Zip42-Cy5; Zip43-Cy5; Zip44-Cy5 Zip24-Cy5; Zip25-Cy5; Zip33-Cy5 Zip34-Cy5; Zip35-Cy5; Zip36-Cy5 Zip29Cy5; Zip30-Cy5; Zip31-Cy5 Zip45-Cy5; Zip46-Cy5; Zip47-Cy5 Zip37-Cy5; Zip40-Cy5; Zip48-Cy5 Zip27-Cy3; Zip28-Cy3; Zip32-Cy3
STEC EIEC, Shigella spp.b Salmonella spp. S. typhi V. cholerae V. mimicus V. furnissii V. fluvialis V. metschnikovii V. parahaemolyticus V. hollisae C. jejuni C. coli C. lari C. upsaliensis C. fetus Y. enterocolitica L. monocytogenes a
LDR products labeled with VeraCode sequence number (Zip) on the 5′-end and the fluorescent dye (Cy3 or-Cy5) labeled on the 3′-end. Up to 3 species-specific LDR products for each genetic target. b EIEC and Shigella spp. share the same genetic targets for identification and are not distinguished from each other.
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2.5. Primer design PCR and LDR primers were designed using Oligo 6.0™ software (Molecular Biology Insights, Cascade, CO, USA). PCR primers were designed based on sequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov) as previously described (Pingle et al. 2007). A list of the genetic targets chosen for primer design is shown in Table 1. A total of 51 PCR primers were designed for all category B bacterial pathogens (Supplemental Table 1). All PCR primers were designed with similar melting temperatures (Tm) to amplify each target gene to ensure accurate identification despite organism-specific gene variations. PCR primers were optimized and validated in uniplex and multiplex reactions for each genetic target. To account for genetic variability within a target genetic sequence, in some instances, multiple PCR primer pairs were generated to amplify a single amplicon. The specificity of the PCR primers in selectively amplifying the desired pathogens was tested in silico by BLAST analysis of the primer sequences against the bacterial sequence databases. Each PCR amplicon was detected by a subsequent LDR reaction. LDR is a linear amplification technique, where 2 adjacent oligonucleotides hybridize
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to a single DNA strand and are ligated by a high fidelity thermostable ligase when there is a perfect match at the junction point. LDR primers were designed to distinguish genus-specific and species-specific single base variants within each PCR amplicon. For example, the Zip25-Cy5 and Zip 24-Cy5 signals are generated from single nucleotide polymorphisms (SNP) that are common to C. jejuni, C. coli, and C. lari (see Table 1). These can be distinguished from each other on the basis of the unique signature generated from the combination of signals (e.g., the unique signature for C. jejuni is “Zip24-Cy5; Zip25-Cy5; Zip38Cy5; Zip39-Cy5; Zip41-Cy5”, whereas that for C. coli is “Zip24-Cy5; Zip25-Cy5; Zip42-Cy5; Zip43-Cy5; Zip44-Cy5”). The upstream LDR primers had melting temperatures of 70 °C and were labeled at the 5′end with a unique VeraCode sequence and C12 amino blocking group. The downstream LDR primers were phosphorylated at the 5′-end, fluorescently labeled at the 3′-end with either a Cy3 dye or Cy5 dye, and had Tm values in the range of 70–75 °C. The 157 LDR primers were optimally partitioned into 2 LDR reactions to prevent primer dimer interactions and ligation deficiency and are shown in Supplemental Table 2. Up to 3 LDR products were designed to detect species-specific single base variants for each genetic target (Table 1). Both PCR and LDR
Fig. 1. Schematic of the PCR/LDR assay with molecular readout using the BeadXpress and Veracode platform. Gene-specific PCR primer pairs were designed to amplify their distinct genetic targets if present. Each PCR amplicon ranged in size from 400 to 600 base pairs. Within each PCR amplicon, we then designed LDR primer pairs to identify single base variants at multiple locations to allow us to distinguish between different pathogenic bacteria. At any given single base variant, the allele specific upstream LDR primers are designed to ligate to the downstream primer only if there is a perfect match at the junction point. The upstream LDR primers bear zipcode complement sequences and amino blocking groups on the 5′end, while the downstream LDR primers have either a Cy3 or Cy5 fluorescent label. Ligation of the LDR primers results in fluorescently labeled products that are subsequently hybridized to zipcode addresses attached to veracode beads. Each bead contains a unique barcode that can be scanned by the BeadXpress reader. Detection of the bead and fluorescent signal scores for the presence of the microbial pathogen.
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primers were synthesized with degenerate bases where required due to sequence variation. PCR and LDR primers were obtained from Integrated DNA Technologies (Coralville, IA, USA).
Hybridization was carried out at 45 °C for 1 h with agitation (1000 rpm). VeraCode Bead Plates were scanned by the BeadXpress Reader System.
2.6. PCR/LDR assay
2.7. Data analysis
A schematic of the PCR/LDR assay is shown in Fig. 1. PCR amplifications were carried out in a total volume of 25 μL containing GeneAmp 1X PCR Buffer II (Applied Biosystems, Foster City, CA, USA), 2.5 mmol/L MgCl2, 200 μmol/L of each dNTP, 5 pmol of each PCR primer, 0.5 ng/nL BSA (Sigma, Saint Louis, MO, USA), 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), and 2 μL of template DNA. Samples were thermocycled using the following parameters: 10 min at 95 °C, followed by 35 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min), and a final extension at 72 °C for 7 min followed by 99.9 °C for 30 min to destroy the polymerase before being held indefinitely at 4 °C. Two separate LDR reactions were prepared containing 500 fmol/μL of each of the appropriate upstream and downstream LDR primers. An aliquot of each primer mix was separately kinased prior to its use in LDR reactions as previously described (Pingle et al. 2007; Rodas et al. 2009; Rondini et al. 2008). LDR reactions were carried out as previously described (Pingle et al. 2007; Rodas et al. 2009; Rondini et al. 2008). Following the LDR reactions, the products were treated with 3′-5′ and 5′-3′ exonucleases to destroy unligated LDR primers. Exonuclease reactions were performed in a total volume of 30 μL containing 1 × 10 4 units of exonuclease I, 2.5 × 10 3 units of Lambda Exonuclease, 1X exonuclease I buffer, and 20 μL of LDR products. Samples were thermocycled using the following parameters: 37 °C for 60 min followed by 80 °C for 10 min before being held indefinitely at 4 °C. VeraCode microbeads (Illumina, San Diego, CA, USA) were prepared according to the manufacturer’s instructions. A total of 48 VeraCode microbeads were mixed and distributed into 96-well microplates using the VeraCode Bead Kitting System in 70% ethanol. Following aspiration of ethanol, 150 μL of 1× saline sodium citrate (SSC) and 0.05% Tween 20 was added to each well. Approximately 100 μL of the buffer was aspirated using the 8-pin aspirator manifold. A total of 30 μL of the LDR products was added to each well.
Signal intensity data obtained from the BeadXpress Reader System were analyzed on Excel spreadsheets. The data were normalized using the median signal values of the negative controls. Signal intensities ≥3-fold higher than the background were considered positive. All samples were tested by the PCR/ LDR assay in quadruplicate. An LDR reaction was considered to be positive when 2 or more of the replicates were positive. Positivity for at least 2 different LDR targets was required for an organism identification. 2.8. Resolution of discordant results Specimens that were PCR/LDR-negative but culture-positive or seeded with a known organism were subjected to repeat DNA extraction and repeat PCR/LDR assay. PCR products were also analyzed by 2% agarose gel electrophoresis to determine if falsenegative PCR/LDR results were due to failure of the PCR step or the LDR step. Culture-positive specimens in which PCR/LDR detected an additional organism were subjected to repeat DNA extraction, repeat PCR/LDR assay, and DNA sequencing. Seeded specimens that were PCR/LDR-positive for an organism that had not been seeded were subjected to repeat DNA extraction, repeat PCR/LDR assay, and DNA sequencing from their corresponding unseeded original specimens. 2.9. Sequencing of discordant samples DNA extracted from discordant samples was subjected to sequencing using the same PCR primers designed for the PCR/LDR assay. PCR reactions were carried out as described above except that a single amplicon was generated in each PCR by using only 1 pair of amplification primers. The PCR products were purified using QIAQuick PCR purification plates (Qiagen) according to the
Table 2 Diagnostic sensitivity and specificity of the PCR/LDR assay on seeded clinical specimens. Organism (no. of seeded specimens/no. of strains) C. jejuni (36/2) Shigella spp. (85/11) Salmonella spp. (165/24) S. typhi (90/7) Y. enterocolitica (29/6) L. monocytogenes (214/22) V. fluvialis (11/1) V. parahaemolyticus (21/1) V. furnissii (21/1) V. mimicus (18/1) V. metschnikovii (22/1) C. coli (63/2) C. lari (21/2) V. cholerae (4/1) EIEC (30/2) STEC (62/7) ETEC (33/2) DAEC (21/1) EPEC (12/3) a b c d
Specimens seeded with listed organism PCR/LDR-posa
PCR/LDR-nega
36 83 163 88 29 206 11 21 21 18 22 63 19 4 30 60 33 21 12
0 2 2 2 0 8 0 0 0 0 0 0 2 0 0 2 0 0 0
Sensitivity (%)
100 97.6 98.8 97.8 100 96.3 100 100 100 100 100 100 90.5 100 100 96.8 100 100 100
Specimens not seeded with listed organismb PCR/LDR-posa
PCR/LDR-nega
0 0 0 0 8 (2)c 1 0 4 (1)c 4 0 0 3 (1)c 1 (1)c 0 4 6 (1)c 1 (1)c 16 (10)c 4
1006 957 877 952 1006 835 1031 1018 1017 1024 1020 978 1020 1038 1008 976 1008 1005 1026
Specificity (%)
100 100 100 100 99.4d 99.9 100 99.7d 99.6 100 100 99.7d 100d 100 99.6 99.5d 100d 99.4d 99.6
PCR/LDR positive or negative for the specified organism. Includes stool specimens that were seeded with another organism and 84 stool specimens that were not seeded. Number in parentheses indicates the number of specimens in which the same organism was detected in the underlying unspiked specimen by PCR and sequencing. Specificity was calculated based on the number of unconfirmed positives.
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manufacturer’s instructions. PCR products were inspected for purity by electrophoresis on 2% agarose gels as well as by measuring the absorbance at 260 and 280 nm. The purified PCR products were adjusted to concentrations of 3–5 ng/μL and sent to Macrogen, Inc. (Rockville, MD, USA) for sequencing. The sequence data obtained were queried against the GenBank database using BLAST (http://www.ncbi.nlm.nih. gov/BLAST). The highest scoring matches were recorded for each organism. 3. Results 3.1. Analytical sensitivity and specificity of the PCR/LDR assay with clinical isolates The analytical specificity of the assay was 100% for pure cultures of all 97 clinical isolates. There were no false positives when testing each pure culture isolate with the PCR/LDR assay, indicating no crossreaction between the 51 PCR primers and 157 LDR primers. We determined the limit of detection of our assay on seeded specimens to range from 10 1 CFU/mL to 10 5 CFU/mL. The concentration in which all pathogens were detected was 10 5 CFU/mL. 3.2. Diagnostic sensitivity and specificity of PCR/LDR with seeded specimens The diagnostic sensitivity and specificity of the PCR/LDR assay were assessed blindly on a large collection of 958 stool specimens that were culture-negative for C. jejuni, Salmonella spp., and Shigella spp. Specimens were individually spiked with 1 × 10 5 CFU/mL of category B agents and then tested by PCR/LDR. An additional 84 culturenegative stool specimens were tested without spiking. The results are shown in Table 2. C. jejuni, Salmonella spp., and Shigella spp. were detected with an overall sensitivity of 98.8% and a specificity of 100% (Table 2). There were 6 false-negative results (Shigella spp., n = 2; Salmonella spp., n = 2; S. typhi, n = 2). Agarose gel electrophoresis revealed an absence of PCR products in these reactions indicating that PCR inhibition was the probable cause for these false-negative results. Stool specimens are known to have high concentrations of PCR inhibitors, such as complex polysaccharides and carbohydrates, heme groups, bile acids, and bilirubins (Monteiro et al. 1997; Nechvatal et al. 2008). The PCR/LDR assay gave similar results for the remaining category B agents including other Campylobacter spp., Vibrio spp., Yersinia enterocolitica, L. monocytogenes, and pathogenic E. coli (EIEC, STEC, ETEC, DAEC, and EPEC); however, there were more discrepant results. The overall sensitivity for these organisms was 97.9% (for 12/15 species the sensitivity was 100%). The assay was unable to detect the seeded organisms in 12 specimens (L. monocytogenes, n = 8; C. lari, n = 2; and STEC, n = 2). PCR/LDR was repeated on these samples and was negative. The absence of PCR products for each false-negative result again suggested that PCR inhibition was the probable cause. In 52 of the seeded specimens, PCR/LDR detected a second organism in addition to the seeded organism. Six of these discordant results involved detection of an organism that had been seeded into an adjacent well within a 96-well microplate. These presumably represent potential cross-contamination or carryover events. The remaining 46 discordant samples were subjected to repeat DNA extraction, repeat PCR/LDR assay, and DNA sequencing from their corresponding unseeded original specimens. In 17 of these samples, the second organism was detected in the original unseeded specimen by both PCR/LDR and sequencing. These specimens contained DAEC (n = 10), Y. enterocolitica (n = 2), STEC (n = 1), ETEC (n = 1), V. parahaemolyticus (n = 1), C. lari (n = 1), and C. coli (n = 1). In the other 29 discordant samples, the second organism was not detected in the unseeded specimen. This may have been caused by crosscontamination or carryover events but may also have been a
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Table 3 Culture-positive samples analyzed by PCR/LDR. Source (type) of specimens
No. of samples
GHESKIO (stool, n = 25; rectal swab, 40 n = 15) WCMC (stool) 1 WCMC (stool) WCMC (stool)
2 1
Culture result
PCR/LDR result (no.)
V. cholerae
V. cholerae (39)a
Salmonella spp. C. jejuni STEC
Negative C. jejuni (2) STEC (1)
a In 11 of these samples, PCR/LDR detected a mixed infection [DAEC (7), EIEC/Shigella spp. (1), V. furnissii (2), and V. mimicus (1)]. The presence of mixed infections was confirmed by sequencing or species-specific PCR.
consequence of prolonged storage of the specimens at 4 °C potentially resulting in DNA degradation. Based on the above analysis, the apparent specificity of PCR/LDR ranged from 99.4% to 100% (Table 2). 3.3. Application of the PCR/LDR assay on culture-positive clinical samples A total of 44 culture-positive clinical specimens were analyzed by the PCR/LDR assay. The results are shown in Table 3. Pathogen identification by the PCR/LDR assay was concordant in 42 of the 44 positive specimens for an overall sensitivity of 95.5%. Of the 44 culture-positive specimens, 1 rectal swab specimen was negative for V. cholerae, and 1 culture-positive stool specimen for Salmonella spp. was negative by the PCR/LDR assay. Repeat PCR/LDR of these 2 samples was also negative. Enrichment of the rectal swab specimen in alkaline peptone water prior to culture on thiosulfate citrate bile salts sucrose (TCBS) agar at 35 °C for 48 h could not recover V. cholerae. The specimen positive for Salmonella spp. was subjected to culture on blood agar and MacConkey agar plates at 35 °C for 48 h; no growth was observed. In 11 of the 40 culture-positive specimens for V. cholerae, PCR/LDR detected a second pathogen (DAEC, n = 7; EIEC/ Shigella spp., n = 1; V. furnissii, n = 2; and V. mimicus, n = 1). The presence of DAEC and EIEC/Shigella spp. was confirmed by sequencing. Because the genetic target to detect all Vibrio pathogens is communal, we were unable to confirm the results via sequencing due to the potential of only the more abundant genetic target being detected. Therefore, we designed species-specific PCR probes to target single base variants within the rpo gene to distinguish the various Vibrio pathogens. The presence of additional pathogens for these 3 specimens was confirmed by species-specific PCR. 4. Discussion In this study, we describe the development and application of a multiplex PCR/LDR assay for the identification of all NIAID category B bacterial food and water-borne pathogens. The assay was designed to PCR amplify 17 genetic targets attributed to a panel of 29 pathogenic bacterial species. Species-specific single base variants within these PCR amplicons are then queried using LDR primer pairs that generate ligation products that are subsequently hybridized to their discrete individual VeraCode beads for identification (Fig. 1). The LDR products produced signals based on bead hybridization and fluorescence and the combination of signals generated a unique molecular signature for each bacterial pathogen. The PCR/LDR assay was initially optimized with DNA extracted from variants of each pathogen isolated from stool or rectal swabs. A number of the pathogens in our panel are rarely encountered. Therefore, we were limited in our ability to obtain isolates or culture-positive clinical specimens for the following organisms: C. fetus, C. upsaliensis, V. hollisae, and EAEC. However, the PCR/LDR assay includes primers to detect and identify all category B bacterial pathogens, including the aforementioned 4 pathogens. A total of 97 clinical isolates representing the remaining category B pathogens were correctly identified with no cross-reactivity.
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The analytical sensitivity of our assay was determined using seeded stool specimens. All organisms were detected at concentration of 10 5 CFU/mL, consistent with other molecular assays for gastrointestinal pathogens (Liu et al. 2013). The diagnostic sensitivity and specificity of the PCR/LDR assay were assessed with culture-negative stool specimens seeded with 1 × 10 5 CFU/mL of category B pathogens. This approach was taken because the positivity rate for pathogens in stool cultures at most academic medical centers in the United States (including NYPH/WCMC) is less than 5%. A large number of separate stool specimens were used in the seeding experiments to assess the robustness of the assay with clinical samples. The sensitivity of PCR/LDR for detecting category B bacteria pathogens ranged from 90% to 100% as determined with a large collection of seeded specimens and a smaller set of specimens from Haiti that contained V. cholerae. The majority of the false-negative reactions may have been caused by PCR inhibitors. Future versions of the assay will include PCR/LDR targets that can serve as an inhibitor control. The apparent specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. The specificity was 100% for C. jejuni, Salmonella spp., and Shigella spp., organisms for which routine stool cultures have been optimized. The putative false-positive PCR/LDR results were restricted to organisms for which selective culture methods are not routinely performed (Y. enterocolitica, L. monocytogenes, and V. parahaemolyticus) or that cannot be detected with standard stool culture techniques (EIEC, DAEC, STEC, and EPEC). Other investigators have seen similar discrepancies when multiplex molecular assays and culture methods are performed on clinical samples. Liu et al. 2013 evaluated the ability of a TaqMan Array Card (TAC) assay to detect a variety of enteric pathogens present in clinical specimens from Tanzania and Bangladesh. The specificity of TAC for detecting Salmonella, V. cholerae, Shigella, and pathogenic E. coli was 71% when compared to culture-based methods but increased to 95% when compared to a PCR-Luminex amplification assay (the corresponding sensitivity values were 99% and 98%, respectively). Due to the extensive and substantial number of enteric bacterial pathogens that can be incriminated as causative agents in stool specimens from infected individuals, the etiological significance of enteric bacteria prompts the need for an assay that can simultaneously detect and identify a large comprehensive panel of pathogens. The widespread and all-inclusive adeptness of the PCR/LDR assay can have a major clinical impact, especially in the design of diagnostic algorithms and the interpretation of epidemiological studies. The PCR/LDR assay is capable of screening a large panel of pathogens to carry out such an investigation, although additional characterization studies of the PCR/LDR assay need to be carried out on the potential interference of various pathogenic loads in mixed infections. Implementation of this molecular assay affords the capability of screening a comprehensive panel of enteric pathogens with enhanced sensitivity and speed of diagnosis, providing a sampleto-result answer in less than 8 hours with an overall diagnostic sensitivity of N98%. Additional applications of PCR/LDR with a universal array readout integrated in a microfluidic device exhibited detection of pathogenic bacteria in b40 min with minimal operator intervention, revealing the potential of PCR/LDR-based molecular analysis systems to generate results rapidly in a fully automated process (Chen et al. 2012). Combining these modular approaches will provide an earlier warning of potential public health threats, as well as a more accurate surveillance and profile of food and water-borne pathogens. This multiplex molecular assay can yield accurate, timely information, resulting in improved patient care while also providing epidemiological data for infection control and virulence/toxin surveillance. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2014.02.022.
Acknowledgments We thank Stephen Jenkins, Audrey Schuetz, and the technical staff of the clinical microbiology laboratory at NYPH/WCMC for collecting and providing clinical isolates and specimens and Bill Pape at GHESKIO for his advice and support. We thank Jianmin Huang for providing the AK16D ligase enzyme. We acknowledge Lee Riley of the University of California Berkeley and the New York City Department of Health for providing us with clinical isolates.
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