Accepted Manuscript Title: Multiplexed detection of foodborne pathogens based on magnetic particles Author: Delfina Brand˜ao Susana Li´ebana Mar´ıa Isabel Pividori PII: DOI: Reference:
S1871-6784(15)00053-9 http://dx.doi.org/doi:10.1016/j.nbt.2015.03.011 NBT 776
To appear in: Received date: Revised date: Accepted date:
2-10-2014 16-3-2015 22-3-2015
Please cite this article as: Brand˜ao, D., Li´ebana, S., Pividori, M.I.,Multiplexed detection of foodborne pathogens based on magnetic particles, New Biotechnology (2015), http://dx.doi.org/10.1016/j.nbt.2015.03.011 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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Multiplexed detection of foodborne pathogens based on magnetic particles
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Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de
Applied Enzyme Technology Ltd., Gwent Group Ltd., Monmouth House, Mamhilad Park, Pontypool, NP4 OHZ, UK.
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Barcelona, 08193 Cerdanyola del Vallès (Bellaterra), Spain
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Delfina Brandão1, Susana Liébana2 and María Isabel Pividori1*
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* Tel: +34 93 581 4937, Fax: +34 93 581 2473. E-mail address:
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*Authors to whom correspondence should be sent: [email protected]
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Abstract
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This paper addresses the novel approaches for the multiplex detection of food poisoning
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bacteria, paying closer attention to three of the most common pathogens involved in
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food outbreaks: Salmonella enterica, Escherichia coli O157:H7 and Listeria
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monocytogenes. End-point and real-time PCR, classical immunological techniques,
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biosensors, microarrays and microfluidic platforms, as well as commercial kits for
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multiplex detection of food pathogens will be reviewed, with special focus on the role
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of magnetic particles in these approaches. Although the immunomagnetic separation for
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capturing single bacteria from contaminating microflora and interfering food
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components has demonstrated to improve the performance on these approaches, the
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integration of magnetic particles for multiplex detection of bacteria is still in a
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preliminary stage and requires further studies.
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Keywords: Food safety, simultaneous detection, magnetic particle, electrochemical
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biosensor, multiplex PCR, food pathogens.
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Introduction
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Food safety in Europe
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Food contamination caused by pathogens is a significant public health concern for
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consumers worldwide. Therefore, identification and detection of microorganisms in
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food processing play an important role at control programs provided by Food regulatory
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agencies, in order to prevent food outbreaks [1, 2]. In 2013, it was reported by the Rapid
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Alert System for Food and Feed (RASFF) that pathogenic microorganisms, mycotoxins
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and pesticide residues were the main cause of notification in Europe, especially for meat
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products, as well as for fruits and vegetables [3]. For instance, the notifications of
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pathogens in meat products are shown in Figure 1, being Salmonella, E. coli and L.
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monocytogenes the most common reported pathogens.
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Hence, food safety standards, such as RASFF were introduced in Europe in order to
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prevent foodborne outbreaks and to ensure that marketed products are safe to the
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consumers.
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Preventive approaches like Hazard Analysis and Critical Control Point (HACCP) and
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the Codex Alimentarius were also successfully implemented, which can considerably
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reduce the survival of pathogens during the process of handling, preparation and storage
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of food. But there is still a need for the development of new tools and technologies to
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prevent problems related to food safety, including rapid approaches for the
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identification and quantification of foodborne pathogens [8, 9].
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Traditional methods for the detection of foodborne pathogens
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Threshold limits for the presence of certain microorganisms had been set down for some
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food products. If the food product contains an amount above the required legislation, it
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must be rejected from the market. Therefore, in this section, the traditional methods
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commonly used for the single detection of foodborne pathogens will be discussed, with
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special focus on their advantages and disadvantages.
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Conventional microbiological culture techniques
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Conventional microbiological culture techniques are currently the gold standard for
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isolation, detection, and identification of microorganisms. These methods are very
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simple, consisting in the following steps: pre-enrichment, selective enrichment,
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selective plating, biochemical screening and serological confirmation. Although they
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are considered to be reliable, they are also time-consuming, laborious and might
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introduce sampling and enumeration errors, due to the low concentration of pathogenic
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bacteria in food samples [9, 10].
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Immunological assays
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Immunological assays (IAs) relies on the specificity of the antigen-antibody
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recognition, being suitable for the detection of whole bacterial cells or specific cellular
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components as lipopolysaccharides or other biomolecules present on the bacterial outer
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membrane. Enzyme Linked ImmunoSorbent Assays (ELISAs), such as sandwich with
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direct and indirect labelling are the most common formats used for the detection of
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pathogens. ELISA methods have been approved by regulatory agencies, being
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commercially available. The limit of detection (LOD) for pathogens are normally in the
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range of 104 and 105 CFU mL-1 and the assay time can take around 48 h, since a pre4
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enrichment step is commonly required in order to achieve the threshold limits for the
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presence of the bacteria on food samples [8- 10].
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Immunomagnetic separation (IMS) is also an example of an immunoassay commonly
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used for bacteria detection. In this technique, superparamagnetic particles modified with
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a variety of molecular groups are coated with antibodies specific to the target bacteria.
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Therefore, bacteria will be captured and concentrated either from a culture medium or
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from complex food matrices. The use of magnetic particles provides several advantages
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to IAs such as (i) pre-concentration of the target bacteria into smaller volumes for
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further testing (ii) reducing and simplifying the pre-enrichment step (iii) eliminating the
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matrix effect of the food components [11].
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Magnetic particles can also be modified with bacteriophages for capturing and pre-
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concentration of bacteria, in an ELISA-like format, named as phagomagnetic
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immunoassay, decreasing significantly the LOD of a classic immunoassay, up to 19
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CFU mL−1 in 2.5 h without any pre-enrichment in milk samples [12].
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Therefore, IAs are advantageous for decreasing the assay reaction time in comparison
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with microbiological culturing techniques, providing also the possibility of being easily
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integrated in automated equipments, which consists an important advantage for
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industrial applications. Nevertheless, the efficiency of an immunoassay is strongly
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dependent on the antibodies affinity and specificity towards the target pathogen. The
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risk of antibody cross-reactions consists of a disadvantage of immunological assays by
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increasing the possibility of false positive results or high background signals [8- 11].
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Nucleic acid amplification methods
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Nucleic acid amplification methods include end-point polymerase chain reaction (PCR)
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and real-time PCR (qPCR) for single or multiplex detection of bacteria. PCR allows the 5
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production of multiple copies of DNA from the amplification of a single copy or a few
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copies of a DNA template. Due to its high sensitivity, nucleic acid amplification has
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been widely used for the identification and detection of pathogens in food samples,
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being considered as an alternative to conventional microbiological culture techniques.
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As occurred in the immunological assays, PCR methodologies require an enrichment
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step, being able to detect, for instance in the case of Salmonella, few CFUs in 25 g of
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food product. The fact that this methodology does not discriminate between live of dead
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cells are pointed out as the main limitations [8- 13].
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Hence, it was shown that traditional methodologies can be sensitive for food
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microbiological control. However, the stricter and increased legislations and controls to
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implement public food safety lead mostly to a need for the development of rapid
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methodologies. In this context, the development of new methodologies with
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multiplexing capabilities becomes an important advantage presenting a cost effective
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and time saving strategy.
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Immunosensors, DNA biosensors and phagosensors
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Over the recent years, a lot of effort has been directed into the study and development of
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rapid methods for foodborne pathogens as an alternative methodology to
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microbiological culturing, IAs and PCR approaches [14]. Biosensors technology has
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been revolutionised research conducted in food safety. These devices are available in a
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wide range of readout platforms, such as surface plasmon resonance [15],
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electrochemical [16] and various kinds of optical biosensors [17]. Most of the current
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developed biosensors for pathogenic bacteria are based on the specific antigen –
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antibody binding reactions, where the antibody is immobilised on the sensor platform to
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capture the bacteria that are of interest. Then, the bacteria detection is measured through
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electrochemical,
optical,
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readouts.
[18]
Moreover,
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biorecognition is also widely used in biosensing, as well as the biorecognition through
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bacteriophages. The integration of magnetic particles in immunosensors, genosensors
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and phagosensors was reported, for instance, in electrochemical based approaches,
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improving the analytical performance in terms of LODs. A magneto immunosensor
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with electrochemical readout was reported for the detection of Salmonella in milk
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(Figure 2, A). In this approach, the bacteria were captured and pre-concentrated from
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milk samples with magnetic micro or nanoparticles through an immunological reaction.
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A second polyclonal antibody labelled with peroxidase was used for the electrochemical
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detection based on a magneto-electrode [16, 19]. This strategy was able to detect 1x104
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CFU mL-1 in 1h. If the sample is pre-enriched for 8 h, as low as 2.7×CFU in 25 g of
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milk were detected accordingly with the legislation. Another approach involves the lysis
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of the captured bacteria after immuno or phagomagnetic separation, followed by
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amplification of the genetic material by PCR with a double-tagging set of primers
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(Figure 2, B). Then, the double-tagged amplicon was immobilised on streptavidin-
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modified magnetic beads based on a high affinity interaction though the biotin tagging
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the 5`end DNA of the amplicon, while the digoxigenin label was used for the enzymatic
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reaction. The electrochemical detection was finally achieved by an enzyme marker,
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such as anti-digoxigenin horseradish peroxidase (HRP). A LOD of 1 CFU mL-1 was
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obtained in 3.5 h without any pre-treatment. If the milk is pre-enriched for 6 h, the
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method is able to feasibly detect as low as 1 CFU in 25 g of milk [20, 21]. The
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integration of the magnetic particles improved the analytical performance of these
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approaches, providing the pre-concentration of the bacteria during the IMS, reducing
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the time required for the pre-enrichment step and the LODs, eliminating the matrix
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effect of the food components and PCR inhibitors, and serving also as a platform for the
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electrochemical readout based on magneto-actuated electrodes.
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Simultaneous detection of pathogenic bacteria
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In this section, different methods for the simultaneous detection of pathogenic bacteria
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based on multiplex PCR, DNA microarrays and biosensors will be fully reviewed and
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summarised in the Tables 1 and 2.
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Multiplex PCR
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End-point multiplex PCR (mPCR) has been widely used to detect simultaneously
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multiple targets in the same amplification reaction. The efficiency of this methodology
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is strongly dependent on different factors, such as primers specificity, buffer,
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magnesium chloride and Taq DNA polymerase concentration, thermal cycling
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conditions and the amount of DNA template. [22] When applied to the detection of
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foodborne pathogenic bacteria, a pre-enrichment step is required in order to enhance
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detection of pathogens in samples. Recently, mPCR for the detection of five different
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pathogens in artificially contaminated pork samples was reported [23- 24]. In Table 1,
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selected studies published in the literature are shown, summarising and highlighting the
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most important parameters in terms of the analytical performance, including the food
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matrix, the time required for the pre-enrichment step, the total assay time and the LODs.
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For instance, the simultaneous detection of E. coli, Salmonella spp. and L.
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monocytogenes by end-point multiplex PCR showed a LOD of 10 CFU in 25 g of
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sample within 15 h of pre-enrichment [25]. The detection by end-point mPCR usually
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requires the analysis of the PCR final products on an agarose gel or on capillary
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electrophoresis-based DNA sequencer, being for this reason more laborious and time
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consuming than Real Time PCR (qPCR), which provides the detection and
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quantification during the amplification process in each cycle. In this methodology, the
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fluorescence intensity of the amplicons is measured by either intercalation of
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fluorescent dyes in the double-stranded DNA or with dual-labelled fluorescent
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oligonucleotide probes, among others readout strategies [26]. The application of qPCR
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for simultaneous detection has been studied for several years and it requires the use
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oligonucleotides tagged with different fluorophores specific for the different
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microorganisms to be detected. Moreover, in some cases an internal amplification
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control (IAC) is recommended to be added into the PCR mixture [26, 27], which
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consists on a non-target DNA sequence, in order to avoid false-negative results caused
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by inhibitors, such as phenolic compounds, fats and glycogen, that may affect different
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steps of the qPCR method. qPCR procedures are in general faster than the related
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conventional methodology, with similar sensitivity. Nevertheless, expensive bench top
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equipment and high technical requirements are the main limitation of this methodology.
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Recently, it has been reported in the literature the combination of magnetic particles
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with PCR based methodologies. As an example, a rapid and simultaneous detection of
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Salmonella, Shigella, and Staphylococcus aureus in fresh pork was reported, being able
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to detect all pathogens at 10 CFU g-1, within 6 h [28, 29]. In this example, the use of
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magnetic particles provided a significant decrease of the total assay time, including the
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pre-enrichment step. This strategy is already commercially available for single
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pathogens detection, proving the potential application of magnetic particles in food
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industry.
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Microfluidic approaches
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Microfluidic systems provide several advantages for the simultaneous determination of
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multiple foodborne bacterial pathogens, such as portability, lower reagent consumption,
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rapidity and possibility for automation. Usually, these systems are combined with
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agarose gel electrophoresis for DNA analysis, but they can also be coupled with other
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platforms [30]. The risk of cross-contamination in the process of sample loading is
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pointed out as one of the main inconvenients of these systems [31]. In the Table 1, some
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approaches for the simultaneous detection of pathogens using PCR based microfluidic
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systems are presented. For instance, the simultaneous detection of S. enterica, E. coli
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and L. monocytogenes was reported within 35 min, with LODs of 399, 314, and 626
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DNA copies per μL, respectively [32].
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DNA microarrays
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DNA microarrays were reported for the detection and identification of several
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pathogens. In these arrays, a DNA probe or oligonucleotide is immobilised at fixed
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positions on a substrate and used to capture the target molecule through hybridisation of
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the amplified DNA, offering in this way higher capacity for multiplexing, as well as the
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possibility of miniaturisation and automation [33]. Several examples of DNA
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microarrays for the simultaneous detection of foodborne pathogens are summarised on
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the Table 1. In this context, it is emphasised that DNA microarrays provide an
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important advantage of bacteria screening in a high number of food samples. The
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detection is achieved by measuring the fluorescence intensity, which presents the
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limitation of being expensive and non-portable. Alternatively, colorimetric methods and
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biochips combined with chemiluminescent labels can be used. Therefore, a DNA
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microarray based on a colorimetric detection was reported using digoxigenin and biotin
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labelled on the DNA. The simultaneous detection of Salmonella spp., Shigella spp., L.
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monocytogenes, and E. coli was achieved with a LOD of 105 CFU mL-1, without any
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enrichment step [34]. A pathogen detection microarray combined with PCR
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methodologies with fluorescence readout was developed for the simultaneous detection
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of E. coli, S. enterica, L. monocytogenes and Campylobacter jejuni being able to detect
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as low as 103 CFU mL-1 of culture medium or food sample [35].
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Combination of DNA microarrays with nanomaterials are also being explored as an
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alternative to overcome problems related to photo bleaching caused by fluorescent the
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organic dyes. In this context, the identification of twelve bacterial strains, using
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quantum dots coated with streptavidin as fluorescent labels was achieved with a LOD of
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10 CFU mL-1 in pure culture, without any enrichment step [36]. Furthermore, magnetic
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particles can also be integrated on DNA microarrays opening the possibility of final
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detection based on a digital camera or a light microscopy [37]. In this context, it was
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reported the detection of E. coli, S. enterica, C. jejuni, presenting LODs of 136, 500,
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and 1 CFU mL-1, respectively, without any enrichment procedures. [38]
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Nevertheless, the microarray fabrication and also the hybridisation procedure can be
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time consuming, presenting the main disadvantage of this methodology.
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Biosensors
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Over the past years, a new challenge has been attracting researchers in this field, the
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design of novel biosensors with multiplexing capabilities, where the integration of
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nanomaterials plays an important role. These novel bionanomaterials, including 11
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nanostructured carbon materials, inorganic nanoparticles (i.e., semi-conducting, noble
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metal and magnetic nanoparticles), among others, appears to be keys in bacteria
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multiplex detection in biosensors, enhancing the biological reactions, providing high
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selectivity and improving the LODs [39, 40, 41, 42].
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In this section, different biosensor strategies will be discussed. These strategies are
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summarised in the Table 2, with special focus on the assay type, detection technique,
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food matrix, LODs and assay times, being classified according to the type of
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nanomaterial integrated in each approach.
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Biosensing based on metallic nanomaterials
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Metallic nanomaterials, such as gold or silver nanoparticles, as well as gold films are
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the most common selection for the immobilisation of biomolecules and/or signal
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amplification. In this context, a biosensor for the detection of Salmonella and E. coli
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based on Raman spectroscopy was reported, in which gold, silver and core–shell
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nanoparticles were coated with a Raman reporter molecule to improve the LOD of the
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assay [43]. This strategy was able to detect both bacteria with a LOD of 102 CFU mL-1
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in 45 min. A multi-channel SPR biosensor for the simultaneous detection of S.
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Typhimurium, L. monocytogenes, C. jejuni, and E. coli based on a sandwich
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immunoassay was also reported, presenting LODs of from 3.4×103 to 1.2×105 CFU mL-
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in 50 min [44].
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Biosensing based on Quantum Dots 12
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Semiconductor particles as quantum dots (QDs) are also a common choice in the
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strategies for multiplexing, either as a support for the target analyte or as a label to
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enhance the optical or electrochemical readout. In this context, an electrochemical
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immunosensor for the multiplex detection of E. coli, Campylobacter and Salmonella
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based on a sandwich immunoassay with QDs modified with three antibodies specific for
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each bacterium was reported with a LOD of 400 CFU mL−1 for Salmonella and
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Campylobacter and 800 CFU mL−1 for E. coli in 1h [45]. Another strategy was reported
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recently using aptamers specific for Vibrio parahaemolyticus and S. Typhimurium
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immobilised on quantum dots. The quantification of bacteria was based on dual
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fluorescence resonance energy transfer (FRET) between QDs and carbon nanoparticles,
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being able to detect 25 and 35 CFU mL-1 of V. parahaemolyticus and S. Typhimurium
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respectively within 2h 20 min. [46]
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Biosensing based on Magnetic Particles
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Along this review, the integration of magnetic particles, either with micrometer or
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nanometer sizes on the strategies for the detection of foodborne pathogenic bacteria has
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been emphasised. The possibility of immobilisation of a variety of biomolecules, such
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as enzymes, antibodies, oligonucleotides onto the magnetic particles surface, as well as
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the possibility of being easily manipulated by an external magnetic field gradient,
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provides a selective capture of the target bacteria, offering an attractive application
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towards the development of new biosensors and microfluidic devices [47].
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Magnetic particles have also been combined with several biosensors platforms,
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especially biosensors with optical readout. In this context, some studies were reported
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using both magnetic particles and quantum dots in a sandwich immunoassay. For one
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side, IMS of bacteria was achieved with further fluorescence detection, using different
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quantum dots modified with antibodies specific for each bacterium [48, 49]. Recently,
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an aptasensor with optical readout for the simultaneous detection of S. aureus, V.
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parahemolyticus, and S. Typhimurium was reported, using magnetic particles and
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multicolor upconversion nanoparticles (UCNPs), as luminescence labels (Figure 3). In
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this strategy, multicolor UCNPs were conjugated with aptamers specific for each
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bacterium and hybridised with the complementary DNA sequence, which was coupled
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to magnetic particles, these last providing an important advantage of improving the
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washing steps. These conjugates are capable of emitting strong visible luminescence
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with the excitation of NIR light (typically 980 nm), using a 980 nm laser, giving three
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independent peaks at different wavelengths for each of the three bacteria. Upon addition
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of the bacteria, these signals are proportionally reduced, since the multicolor UCNPs
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conjugated with the aptamers react with their specific bacterial target being then
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eliminated as a supernatant after applying a magnetic field, as shown in Figure 3. The
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remaining UCNPs-MNPs were then separated and washed three times, and the
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luminescence was measured with a 980 nm excitation laser.
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The concentration of the three bacteria was related to the corresponding emission peak
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of the multicolor UCNPs. The basic principle of the strategy was that aptamers could
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form a defined conformation when binding to the targets and were also able to hybridise
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to the complementary DNA sequences attached to the MPs to form a duplex structure.
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When the targets and the complementary oligonucleotides were introduced, the
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aptamers preferentially bound to the targets, resulting in the specific recognition of the
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targets. Therefore, < 25 CFU mL-1 of all pathogens were detected in approximately 1h
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with this approach [50].
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Electrochemical immuno and genosensors have been extensively explored for food
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safety applications due to their high sensitivity, rapidity, low cost and possibility of
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being a hand-held platform operated by batteries for field applications. However, the
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use of electrochemical biosensors remains in a preliminary stage towards multiplex
328
detection of bacteria [1].
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Commercial approaches
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The commercialisation of novel devices for the detection of food microorganisms is
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based on a set of specific standards in food microbiology, such as quality control of
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culture media, preparation of test samples, uncertainty estimation, method validation
334
and proficiency testing [51].
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Currently, there are an increasing number of kits commercially available for a rapid
336
simple and reliable detection of pathogenic bacteria in food samples. In Table 3,
337
selected kits, mostly for single bacteria detection are compared in terms of assay format,
338
target pathogens, pre-enrichment step time, total assay time and LODs. Commercial kits
339
are based on different assays formats either immunological assays or PCR
340
methodologies for the detection of several foodborne pathogens, being able to detect in
341
general 1 CFU in 25 g of food sample as required for the legislation, after a pre-
342
enrichment culturing step. Most recent kits make use of a fully automated system,
343
which reduces the assay time, increasing the number of samples per test until 300
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samples in one assay (Atlas® System, Assurance® EIA or GeneQuence). Rapid nucleic
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acid amplification and detection technologies have been increasingly applied to
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pathogen detection in food industry. In DuPont™ BAX® System, a qPCR methodology
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is used for the detection of several pathogens with LODs of one CFU per food sample
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ranged from 25 g to 375 g.
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The use of nano-microsized particles on bioassays has been successfully introduced into
350
food industry. Lateral Flow System has been commercialised by Dupont (DuPontTM
351
Lateral Flow System) and Biocontrol (VIP® Gold), where food samples are combined
352
with gold-colloid particles coated with antibodies specific for the target bacteria. The
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results are produced in only 10 min, being for this reason a suitable tool for pathogens
354
screening.
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Finally, magnetic particles modified with antibodies specific to the target bacteria have
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been used for IMS as a pre-concentration tool from food samples, being able to reduce
357
substantially the time of enrichment step. IMS is usually combined with PCR
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methodologies, as occurred in Assurance GDS® MPX and Pathatrix®. Currently, the
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commercialisation of kits for the simultaneous detection of foodborne pathogens is
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available in a reduced number, being mostly based on PCR methodology (Assurance
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GDS® MPX).
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Conclusions
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A considerable progress regarding food safety in EU has been done in terms of rapid
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and multiplexed approaches for detecting bacteria outbreaks. However, food
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contamination caused by pathogenic bacteria is still a serious threat for the consumers.
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The common strategies for the detection of pathogenic microorganisms are consisted of
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the gold standard conventional microbiological culturing techniques, IAs and PCR 16
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methodologies. The development of novel strategies with multiplexing capabilities is
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highlighted as a rapid and cost effective alternative for the detection of bacteria.
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Therefore, in this review, it was shown that mPCR based methodologies could detect
374
below 10 CFU in 25 g of sample after a pre-enrichment step up to 30 h [25, 53]. The
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integration of magnetic particles with PCR based methodologies leads to a LOD of less
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than 10 CFU g-1 within 6 h [28]. DNA microarrays showed LODs below 500 CFU mL-1
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when using magnetic particles, in approximately 3.5 h, without the need of a pre-
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enrichment step [38]. Biosensors combined with nanomaterials have led to a significant
379
improvement of the assay time and LOD. In this context, no significant differences were
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noticed between the different nanomaterials, however it was observed that the
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integration of magnetic particles allows a significant decrease of the enrichment times,
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reducing thus the total assay time [50, 59-60].
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To summarise, in food safety applications the integration of magnetic carriers and IMS
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step for capturing the bacteria through an immunological reaction from contaminating
385
microflora and interfering food components introduces advantages on the analytical
386
performance due to the pre-concentration upon magnetic actuation for further testing or
387
readout. For instance, the matrix effect is eliminated and the PCR inhibitors are avoided
388
which leads to a decrease of the background signals. The improvement of the washing
389
steps and the decrease of the time required for enrichment steps are also other
390
advantages. Finally, magnetic particles offer an attractive support to be incorporated in
391
magnetic actuated biosensors, microfluidic platforms or other devices.
392
Magnetic particles have also been successfully integrated in commercially available kits
393
for a rapid, simple and reliable detection of single pathogenic bacteria. However, there
394
are still only a few commercial kits available for simultaneous detection, with integrated
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magnetic nanoparticle functionality. As a conclusion the integration of magnetic
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particles for the multiplex detection of bacteria is still in a preliminary stage, requiring
397
further studies due to their promising features.
398
Acknowledgments
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Financial support from BioMaX “Novel diagnostic bioassays based on magnetic
401
particles”, Marie Curie Initial Training Networks (FP7-PEOPLE-2010-ITN) and the
402
Ministry of Economy and Competitiveness (MINECO), Madrid (Project BIO2013-
403
41242-R) are acknowledged.
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FIGURE CAPTIONS
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Figure 1. Notifications for pathogens found in meat samples in 2012 and 2013.
407
Figure 2. Integration of magnetic particles in immunosensors and genosensors. After an
408
immunomagnetic separation step, (A) a magneto immunosensor and (B) a magneto
409
genosensor with electrochemical readout were reported for the detection of Salmonella
410
in milk. Adapted from [45].
411
Table 1. Strategies for the simultaneous detection of pathogenic bacteria based on
412
Nucleic acid amplification methods, Multiplex PCR based microfluidic systems and
413
DNA microarrays.
414
Table 2. Biosensors platforms based on the integration of nanomaterials for the
415
simultaneous detection of bacteria.
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Figure 3. Simultaneous detection of S. aureus, V. parahemolyticus, and S.
417
Typhimurium, using magnetic particles (MNPs) and multicolor upconversion
418
nanoparticles (UCNPs), as luminescence labels. UCNPs were modified with aptamers
419
(Apt1-3) specific for the different bacterium. Complementary oligonucleotides (c1-3DNA-
420
MPs) were immobilised on the MNPs. Reprinted with permission from [50]. Copyright
421
(2014) American Chemical Society.
422
Table 3. Commercial kits for the detection of pathogenic bacteria.
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Table 1 Preenrichment
Total assay time
LOD
Ref.
Agarose gel
artificially contaminated pork samples
overnight
~ 1h 40 min
9-670 CFU mL-1
[23]
Agarose gel
artificially contaminated meat samples
24 h
~ 1h 35 min
10–17 CFU g1 sample
[24]
>15 h
~ 1h 45 min
30h
1h 10 min
qPCR for Salmonella spp., E. coli, L. monocytogenes
Fluorescence
qPCR for Salmonella spp., E. coli, L. monocytogenes
Fluorescence
Nonaplex qPCR for L. monocytogenes, Campylobacter, Salmonella, enteropathogenic E. coli
Fluorescence
IMS-qPCR assay for Salmonella spp., Shigella spp., S. aureus
Fluorescence
IMS -mPCR assay for S. Typhimurium, E. coli, L. monocytogenes
Agarose gel
agarose gel electrophoresi s and GoldView™ agarose gel stained with GoldView™
Oscillatory-flow multiplex PCR PCR for S. enterica, E. coli, L. monocytogenes
Microchip capillary electrophoresis for V. parahemolyticus, Salmonella, E. coli, Shigella DNA microarray for Salmonella spp., Shigella spp., L. monocytogenes, E. coli
Capillary electrophoresis
Chemilumines -cence
DNA microarray for E. coli, S. enterica, L. monocytogenes, C. jejuni
Fluorescence
DNA microarray for 12 different bacterial strains using QDs
Fluorescence
[25]
1 CFU in 25 g sample
[52]
30h
55 min
5 CFU in 25 g sample
[53]
20 h
60 min
S1871-6784(15)00053-9 http://dx.doi.org/doi:10.1016/j.nbt.2015.03.011 NBT 776
To appear in: Received date: Revised date: Accepted date:
2-10-2014 16-3-2015 22-3-2015
Please cite this article as: Brand˜ao, D., Li´ebana, S., Pividori, M.I.,Multiplexed detection of foodborne pathogens based on magnetic particles, New Biotechnology (2015), http://dx.doi.org/10.1016/j.nbt.2015.03.011 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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Multiplexed detection of foodborne pathogens based on magnetic particles
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Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de
Applied Enzyme Technology Ltd., Gwent Group Ltd., Monmouth House, Mamhilad Park, Pontypool, NP4 OHZ, UK.
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Barcelona, 08193 Cerdanyola del Vallès (Bellaterra), Spain
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Delfina Brandão1, Susana Liébana2 and María Isabel Pividori1*
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* Tel: +34 93 581 4937, Fax: +34 93 581 2473. E-mail address:
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*Authors to whom correspondence should be sent: [email protected]
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Abstract
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This paper addresses the novel approaches for the multiplex detection of food poisoning
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bacteria, paying closer attention to three of the most common pathogens involved in
17
food outbreaks: Salmonella enterica, Escherichia coli O157:H7 and Listeria
18
monocytogenes. End-point and real-time PCR, classical immunological techniques,
19
biosensors, microarrays and microfluidic platforms, as well as commercial kits for
20
multiplex detection of food pathogens will be reviewed, with special focus on the role
21
of magnetic particles in these approaches. Although the immunomagnetic separation for
22
capturing single bacteria from contaminating microflora and interfering food
23
components has demonstrated to improve the performance on these approaches, the
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integration of magnetic particles for multiplex detection of bacteria is still in a
25
preliminary stage and requires further studies.
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Keywords: Food safety, simultaneous detection, magnetic particle, electrochemical
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biosensor, multiplex PCR, food pathogens.
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Introduction
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Food safety in Europe
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Food contamination caused by pathogens is a significant public health concern for
31
consumers worldwide. Therefore, identification and detection of microorganisms in
32
food processing play an important role at control programs provided by Food regulatory
33
agencies, in order to prevent food outbreaks [1, 2]. In 2013, it was reported by the Rapid
34
Alert System for Food and Feed (RASFF) that pathogenic microorganisms, mycotoxins
35
and pesticide residues were the main cause of notification in Europe, especially for meat
36
products, as well as for fruits and vegetables [3]. For instance, the notifications of
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pathogens in meat products are shown in Figure 1, being Salmonella, E. coli and L.
38
monocytogenes the most common reported pathogens.
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Preferred position for Figure 1
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Hence, food safety standards, such as RASFF were introduced in Europe in order to
43
prevent foodborne outbreaks and to ensure that marketed products are safe to the
44
consumers.
45
Preventive approaches like Hazard Analysis and Critical Control Point (HACCP) and
46
the Codex Alimentarius were also successfully implemented, which can considerably
47
reduce the survival of pathogens during the process of handling, preparation and storage
48
of food. But there is still a need for the development of new tools and technologies to
49
prevent problems related to food safety, including rapid approaches for the
50
identification and quantification of foodborne pathogens [8, 9].
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Traditional methods for the detection of foodborne pathogens
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Threshold limits for the presence of certain microorganisms had been set down for some
54
food products. If the food product contains an amount above the required legislation, it
55
must be rejected from the market. Therefore, in this section, the traditional methods
56
commonly used for the single detection of foodborne pathogens will be discussed, with
57
special focus on their advantages and disadvantages.
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Conventional microbiological culture techniques
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Conventional microbiological culture techniques are currently the gold standard for
61
isolation, detection, and identification of microorganisms. These methods are very
62
simple, consisting in the following steps: pre-enrichment, selective enrichment,
63
selective plating, biochemical screening and serological confirmation. Although they
64
are considered to be reliable, they are also time-consuming, laborious and might
65
introduce sampling and enumeration errors, due to the low concentration of pathogenic
66
bacteria in food samples [9, 10].
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Immunological assays
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Immunological assays (IAs) relies on the specificity of the antigen-antibody
70
recognition, being suitable for the detection of whole bacterial cells or specific cellular
71
components as lipopolysaccharides or other biomolecules present on the bacterial outer
72
membrane. Enzyme Linked ImmunoSorbent Assays (ELISAs), such as sandwich with
73
direct and indirect labelling are the most common formats used for the detection of
74
pathogens. ELISA methods have been approved by regulatory agencies, being
75
commercially available. The limit of detection (LOD) for pathogens are normally in the
76
range of 104 and 105 CFU mL-1 and the assay time can take around 48 h, since a pre4
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enrichment step is commonly required in order to achieve the threshold limits for the
78
presence of the bacteria on food samples [8- 10].
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Immunomagnetic separation (IMS) is also an example of an immunoassay commonly
80
used for bacteria detection. In this technique, superparamagnetic particles modified with
81
a variety of molecular groups are coated with antibodies specific to the target bacteria.
82
Therefore, bacteria will be captured and concentrated either from a culture medium or
83
from complex food matrices. The use of magnetic particles provides several advantages
84
to IAs such as (i) pre-concentration of the target bacteria into smaller volumes for
85
further testing (ii) reducing and simplifying the pre-enrichment step (iii) eliminating the
86
matrix effect of the food components [11].
87
Magnetic particles can also be modified with bacteriophages for capturing and pre-
88
concentration of bacteria, in an ELISA-like format, named as phagomagnetic
89
immunoassay, decreasing significantly the LOD of a classic immunoassay, up to 19
90
CFU mL−1 in 2.5 h without any pre-enrichment in milk samples [12].
91
Therefore, IAs are advantageous for decreasing the assay reaction time in comparison
92
with microbiological culturing techniques, providing also the possibility of being easily
93
integrated in automated equipments, which consists an important advantage for
94
industrial applications. Nevertheless, the efficiency of an immunoassay is strongly
95
dependent on the antibodies affinity and specificity towards the target pathogen. The
96
risk of antibody cross-reactions consists of a disadvantage of immunological assays by
97
increasing the possibility of false positive results or high background signals [8- 11].
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Nucleic acid amplification methods
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Nucleic acid amplification methods include end-point polymerase chain reaction (PCR)
101
and real-time PCR (qPCR) for single or multiplex detection of bacteria. PCR allows the 5
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production of multiple copies of DNA from the amplification of a single copy or a few
103
copies of a DNA template. Due to its high sensitivity, nucleic acid amplification has
104
been widely used for the identification and detection of pathogens in food samples,
105
being considered as an alternative to conventional microbiological culture techniques.
106
As occurred in the immunological assays, PCR methodologies require an enrichment
107
step, being able to detect, for instance in the case of Salmonella, few CFUs in 25 g of
108
food product. The fact that this methodology does not discriminate between live of dead
109
cells are pointed out as the main limitations [8- 13].
110
Hence, it was shown that traditional methodologies can be sensitive for food
111
microbiological control. However, the stricter and increased legislations and controls to
112
implement public food safety lead mostly to a need for the development of rapid
113
methodologies. In this context, the development of new methodologies with
114
multiplexing capabilities becomes an important advantage presenting a cost effective
115
and time saving strategy.
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Immunosensors, DNA biosensors and phagosensors
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Over the recent years, a lot of effort has been directed into the study and development of
119
rapid methods for foodborne pathogens as an alternative methodology to
120
microbiological culturing, IAs and PCR approaches [14]. Biosensors technology has
121
been revolutionised research conducted in food safety. These devices are available in a
122
wide range of readout platforms, such as surface plasmon resonance [15],
123
electrochemical [16] and various kinds of optical biosensors [17]. Most of the current
124
developed biosensors for pathogenic bacteria are based on the specific antigen –
125
antibody binding reactions, where the antibody is immobilised on the sensor platform to
126
capture the bacteria that are of interest. Then, the bacteria detection is measured through
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electrochemical,
optical,
or
piezoelectric
readouts.
[18]
Moreover,
genetic
128
biorecognition is also widely used in biosensing, as well as the biorecognition through
129
bacteriophages. The integration of magnetic particles in immunosensors, genosensors
130
and phagosensors was reported, for instance, in electrochemical based approaches,
131
improving the analytical performance in terms of LODs. A magneto immunosensor
132
with electrochemical readout was reported for the detection of Salmonella in milk
133
(Figure 2, A). In this approach, the bacteria were captured and pre-concentrated from
134
milk samples with magnetic micro or nanoparticles through an immunological reaction.
135
A second polyclonal antibody labelled with peroxidase was used for the electrochemical
136
detection based on a magneto-electrode [16, 19]. This strategy was able to detect 1x104
137
CFU mL-1 in 1h. If the sample is pre-enriched for 8 h, as low as 2.7×CFU in 25 g of
138
milk were detected accordingly with the legislation. Another approach involves the lysis
139
of the captured bacteria after immuno or phagomagnetic separation, followed by
140
amplification of the genetic material by PCR with a double-tagging set of primers
141
(Figure 2, B). Then, the double-tagged amplicon was immobilised on streptavidin-
142
modified magnetic beads based on a high affinity interaction though the biotin tagging
143
the 5`end DNA of the amplicon, while the digoxigenin label was used for the enzymatic
144
reaction. The electrochemical detection was finally achieved by an enzyme marker,
145
such as anti-digoxigenin horseradish peroxidase (HRP). A LOD of 1 CFU mL-1 was
146
obtained in 3.5 h without any pre-treatment. If the milk is pre-enriched for 6 h, the
147
method is able to feasibly detect as low as 1 CFU in 25 g of milk [20, 21]. The
148
integration of the magnetic particles improved the analytical performance of these
149
approaches, providing the pre-concentration of the bacteria during the IMS, reducing
150
the time required for the pre-enrichment step and the LODs, eliminating the matrix
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effect of the food components and PCR inhibitors, and serving also as a platform for the
152
electrochemical readout based on magneto-actuated electrodes.
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Simultaneous detection of pathogenic bacteria
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In this section, different methods for the simultaneous detection of pathogenic bacteria
158
based on multiplex PCR, DNA microarrays and biosensors will be fully reviewed and
159
summarised in the Tables 1 and 2.
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Multiplex PCR
162
End-point multiplex PCR (mPCR) has been widely used to detect simultaneously
163
multiple targets in the same amplification reaction. The efficiency of this methodology
164
is strongly dependent on different factors, such as primers specificity, buffer,
165
magnesium chloride and Taq DNA polymerase concentration, thermal cycling
166
conditions and the amount of DNA template. [22] When applied to the detection of
167
foodborne pathogenic bacteria, a pre-enrichment step is required in order to enhance
168
detection of pathogens in samples. Recently, mPCR for the detection of five different
169
pathogens in artificially contaminated pork samples was reported [23- 24]. In Table 1,
170
selected studies published in the literature are shown, summarising and highlighting the
171
most important parameters in terms of the analytical performance, including the food
172
matrix, the time required for the pre-enrichment step, the total assay time and the LODs.
173
For instance, the simultaneous detection of E. coli, Salmonella spp. and L.
174
monocytogenes by end-point multiplex PCR showed a LOD of 10 CFU in 25 g of
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sample within 15 h of pre-enrichment [25]. The detection by end-point mPCR usually
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requires the analysis of the PCR final products on an agarose gel or on capillary
177
electrophoresis-based DNA sequencer, being for this reason more laborious and time
178
consuming than Real Time PCR (qPCR), which provides the detection and
179
quantification during the amplification process in each cycle. In this methodology, the
180
fluorescence intensity of the amplicons is measured by either intercalation of
181
fluorescent dyes in the double-stranded DNA or with dual-labelled fluorescent
182
oligonucleotide probes, among others readout strategies [26]. The application of qPCR
183
for simultaneous detection has been studied for several years and it requires the use
184
oligonucleotides tagged with different fluorophores specific for the different
185
microorganisms to be detected. Moreover, in some cases an internal amplification
186
control (IAC) is recommended to be added into the PCR mixture [26, 27], which
187
consists on a non-target DNA sequence, in order to avoid false-negative results caused
188
by inhibitors, such as phenolic compounds, fats and glycogen, that may affect different
189
steps of the qPCR method. qPCR procedures are in general faster than the related
190
conventional methodology, with similar sensitivity. Nevertheless, expensive bench top
191
equipment and high technical requirements are the main limitation of this methodology.
192
Recently, it has been reported in the literature the combination of magnetic particles
193
with PCR based methodologies. As an example, a rapid and simultaneous detection of
194
Salmonella, Shigella, and Staphylococcus aureus in fresh pork was reported, being able
195
to detect all pathogens at 10 CFU g-1, within 6 h [28, 29]. In this example, the use of
196
magnetic particles provided a significant decrease of the total assay time, including the
197
pre-enrichment step. This strategy is already commercially available for single
198
pathogens detection, proving the potential application of magnetic particles in food
199
industry.
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Microfluidic approaches
202
Microfluidic systems provide several advantages for the simultaneous determination of
203
multiple foodborne bacterial pathogens, such as portability, lower reagent consumption,
204
rapidity and possibility for automation. Usually, these systems are combined with
205
agarose gel electrophoresis for DNA analysis, but they can also be coupled with other
206
platforms [30]. The risk of cross-contamination in the process of sample loading is
207
pointed out as one of the main inconvenients of these systems [31]. In the Table 1, some
208
approaches for the simultaneous detection of pathogens using PCR based microfluidic
209
systems are presented. For instance, the simultaneous detection of S. enterica, E. coli
210
and L. monocytogenes was reported within 35 min, with LODs of 399, 314, and 626
211
DNA copies per μL, respectively [32].
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DNA microarrays
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DNA microarrays were reported for the detection and identification of several
217
pathogens. In these arrays, a DNA probe or oligonucleotide is immobilised at fixed
218
positions on a substrate and used to capture the target molecule through hybridisation of
219
the amplified DNA, offering in this way higher capacity for multiplexing, as well as the
220
possibility of miniaturisation and automation [33]. Several examples of DNA
221
microarrays for the simultaneous detection of foodborne pathogens are summarised on
222
the Table 1. In this context, it is emphasised that DNA microarrays provide an
223
important advantage of bacteria screening in a high number of food samples. The
224
detection is achieved by measuring the fluorescence intensity, which presents the
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limitation of being expensive and non-portable. Alternatively, colorimetric methods and
226
biochips combined with chemiluminescent labels can be used. Therefore, a DNA
227
microarray based on a colorimetric detection was reported using digoxigenin and biotin
228
labelled on the DNA. The simultaneous detection of Salmonella spp., Shigella spp., L.
229
monocytogenes, and E. coli was achieved with a LOD of 105 CFU mL-1, without any
230
enrichment step [34]. A pathogen detection microarray combined with PCR
231
methodologies with fluorescence readout was developed for the simultaneous detection
232
of E. coli, S. enterica, L. monocytogenes and Campylobacter jejuni being able to detect
233
as low as 103 CFU mL-1 of culture medium or food sample [35].
234
Combination of DNA microarrays with nanomaterials are also being explored as an
235
alternative to overcome problems related to photo bleaching caused by fluorescent the
236
organic dyes. In this context, the identification of twelve bacterial strains, using
237
quantum dots coated with streptavidin as fluorescent labels was achieved with a LOD of
238
10 CFU mL-1 in pure culture, without any enrichment step [36]. Furthermore, magnetic
239
particles can also be integrated on DNA microarrays opening the possibility of final
240
detection based on a digital camera or a light microscopy [37]. In this context, it was
241
reported the detection of E. coli, S. enterica, C. jejuni, presenting LODs of 136, 500,
242
and 1 CFU mL-1, respectively, without any enrichment procedures. [38]
243
Nevertheless, the microarray fabrication and also the hybridisation procedure can be
244
time consuming, presenting the main disadvantage of this methodology.
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Biosensors
247
Over the past years, a new challenge has been attracting researchers in this field, the
248
design of novel biosensors with multiplexing capabilities, where the integration of
249
nanomaterials plays an important role. These novel bionanomaterials, including 11
Page 11 of 34
nanostructured carbon materials, inorganic nanoparticles (i.e., semi-conducting, noble
251
metal and magnetic nanoparticles), among others, appears to be keys in bacteria
252
multiplex detection in biosensors, enhancing the biological reactions, providing high
253
selectivity and improving the LODs [39, 40, 41, 42].
254
In this section, different biosensor strategies will be discussed. These strategies are
255
summarised in the Table 2, with special focus on the assay type, detection technique,
256
food matrix, LODs and assay times, being classified according to the type of
257
nanomaterial integrated in each approach.
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Biosensing based on metallic nanomaterials
263
Metallic nanomaterials, such as gold or silver nanoparticles, as well as gold films are
264
the most common selection for the immobilisation of biomolecules and/or signal
265
amplification. In this context, a biosensor for the detection of Salmonella and E. coli
266
based on Raman spectroscopy was reported, in which gold, silver and core–shell
267
nanoparticles were coated with a Raman reporter molecule to improve the LOD of the
268
assay [43]. This strategy was able to detect both bacteria with a LOD of 102 CFU mL-1
269
in 45 min. A multi-channel SPR biosensor for the simultaneous detection of S.
270
Typhimurium, L. monocytogenes, C. jejuni, and E. coli based on a sandwich
271
immunoassay was also reported, presenting LODs of from 3.4×103 to 1.2×105 CFU mL-
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in 50 min [44].
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Biosensing based on Quantum Dots 12
Page 12 of 34
Semiconductor particles as quantum dots (QDs) are also a common choice in the
276
strategies for multiplexing, either as a support for the target analyte or as a label to
277
enhance the optical or electrochemical readout. In this context, an electrochemical
278
immunosensor for the multiplex detection of E. coli, Campylobacter and Salmonella
279
based on a sandwich immunoassay with QDs modified with three antibodies specific for
280
each bacterium was reported with a LOD of 400 CFU mL−1 for Salmonella and
281
Campylobacter and 800 CFU mL−1 for E. coli in 1h [45]. Another strategy was reported
282
recently using aptamers specific for Vibrio parahaemolyticus and S. Typhimurium
283
immobilised on quantum dots. The quantification of bacteria was based on dual
284
fluorescence resonance energy transfer (FRET) between QDs and carbon nanoparticles,
285
being able to detect 25 and 35 CFU mL-1 of V. parahaemolyticus and S. Typhimurium
286
respectively within 2h 20 min. [46]
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Biosensing based on Magnetic Particles
289
Along this review, the integration of magnetic particles, either with micrometer or
290
nanometer sizes on the strategies for the detection of foodborne pathogenic bacteria has
291
been emphasised. The possibility of immobilisation of a variety of biomolecules, such
292
as enzymes, antibodies, oligonucleotides onto the magnetic particles surface, as well as
293
the possibility of being easily manipulated by an external magnetic field gradient,
294
provides a selective capture of the target bacteria, offering an attractive application
295
towards the development of new biosensors and microfluidic devices [47].
296
Magnetic particles have also been combined with several biosensors platforms,
297
especially biosensors with optical readout. In this context, some studies were reported
298
using both magnetic particles and quantum dots in a sandwich immunoassay. For one
299
side, IMS of bacteria was achieved with further fluorescence detection, using different
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quantum dots modified with antibodies specific for each bacterium [48, 49]. Recently,
301
an aptasensor with optical readout for the simultaneous detection of S. aureus, V.
302
parahemolyticus, and S. Typhimurium was reported, using magnetic particles and
303
multicolor upconversion nanoparticles (UCNPs), as luminescence labels (Figure 3). In
304
this strategy, multicolor UCNPs were conjugated with aptamers specific for each
305
bacterium and hybridised with the complementary DNA sequence, which was coupled
306
to magnetic particles, these last providing an important advantage of improving the
307
washing steps. These conjugates are capable of emitting strong visible luminescence
308
with the excitation of NIR light (typically 980 nm), using a 980 nm laser, giving three
309
independent peaks at different wavelengths for each of the three bacteria. Upon addition
310
of the bacteria, these signals are proportionally reduced, since the multicolor UCNPs
311
conjugated with the aptamers react with their specific bacterial target being then
312
eliminated as a supernatant after applying a magnetic field, as shown in Figure 3. The
313
remaining UCNPs-MNPs were then separated and washed three times, and the
314
luminescence was measured with a 980 nm excitation laser.
315
The concentration of the three bacteria was related to the corresponding emission peak
316
of the multicolor UCNPs. The basic principle of the strategy was that aptamers could
317
form a defined conformation when binding to the targets and were also able to hybridise
318
to the complementary DNA sequences attached to the MPs to form a duplex structure.
319
When the targets and the complementary oligonucleotides were introduced, the
320
aptamers preferentially bound to the targets, resulting in the specific recognition of the
321
targets. Therefore, < 25 CFU mL-1 of all pathogens were detected in approximately 1h
322
with this approach [50].
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Electrochemical immuno and genosensors have been extensively explored for food
325
safety applications due to their high sensitivity, rapidity, low cost and possibility of
326
being a hand-held platform operated by batteries for field applications. However, the
327
use of electrochemical biosensors remains in a preliminary stage towards multiplex
328
detection of bacteria [1].
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Commercial approaches
331
The commercialisation of novel devices for the detection of food microorganisms is
332
based on a set of specific standards in food microbiology, such as quality control of
333
culture media, preparation of test samples, uncertainty estimation, method validation
334
and proficiency testing [51].
335
Currently, there are an increasing number of kits commercially available for a rapid
336
simple and reliable detection of pathogenic bacteria in food samples. In Table 3,
337
selected kits, mostly for single bacteria detection are compared in terms of assay format,
338
target pathogens, pre-enrichment step time, total assay time and LODs. Commercial kits
339
are based on different assays formats either immunological assays or PCR
340
methodologies for the detection of several foodborne pathogens, being able to detect in
341
general 1 CFU in 25 g of food sample as required for the legislation, after a pre-
342
enrichment culturing step. Most recent kits make use of a fully automated system,
343
which reduces the assay time, increasing the number of samples per test until 300
344
samples in one assay (Atlas® System, Assurance® EIA or GeneQuence). Rapid nucleic
345
acid amplification and detection technologies have been increasingly applied to
346
pathogen detection in food industry. In DuPont™ BAX® System, a qPCR methodology
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Page 15 of 34
is used for the detection of several pathogens with LODs of one CFU per food sample
348
ranged from 25 g to 375 g.
349
The use of nano-microsized particles on bioassays has been successfully introduced into
350
food industry. Lateral Flow System has been commercialised by Dupont (DuPontTM
351
Lateral Flow System) and Biocontrol (VIP® Gold), where food samples are combined
352
with gold-colloid particles coated with antibodies specific for the target bacteria. The
353
results are produced in only 10 min, being for this reason a suitable tool for pathogens
354
screening.
355
Finally, magnetic particles modified with antibodies specific to the target bacteria have
356
been used for IMS as a pre-concentration tool from food samples, being able to reduce
357
substantially the time of enrichment step. IMS is usually combined with PCR
358
methodologies, as occurred in Assurance GDS® MPX and Pathatrix®. Currently, the
359
commercialisation of kits for the simultaneous detection of foodborne pathogens is
360
available in a reduced number, being mostly based on PCR methodology (Assurance
361
GDS® MPX).
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Preferred position for Table 3
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365
Conclusions
366
A considerable progress regarding food safety in EU has been done in terms of rapid
367
and multiplexed approaches for detecting bacteria outbreaks. However, food
368
contamination caused by pathogenic bacteria is still a serious threat for the consumers.
369
The common strategies for the detection of pathogenic microorganisms are consisted of
370
the gold standard conventional microbiological culturing techniques, IAs and PCR 16
Page 16 of 34
methodologies. The development of novel strategies with multiplexing capabilities is
372
highlighted as a rapid and cost effective alternative for the detection of bacteria.
373
Therefore, in this review, it was shown that mPCR based methodologies could detect
374
below 10 CFU in 25 g of sample after a pre-enrichment step up to 30 h [25, 53]. The
375
integration of magnetic particles with PCR based methodologies leads to a LOD of less
376
than 10 CFU g-1 within 6 h [28]. DNA microarrays showed LODs below 500 CFU mL-1
377
when using magnetic particles, in approximately 3.5 h, without the need of a pre-
378
enrichment step [38]. Biosensors combined with nanomaterials have led to a significant
379
improvement of the assay time and LOD. In this context, no significant differences were
380
noticed between the different nanomaterials, however it was observed that the
381
integration of magnetic particles allows a significant decrease of the enrichment times,
382
reducing thus the total assay time [50, 59-60].
383
To summarise, in food safety applications the integration of magnetic carriers and IMS
384
step for capturing the bacteria through an immunological reaction from contaminating
385
microflora and interfering food components introduces advantages on the analytical
386
performance due to the pre-concentration upon magnetic actuation for further testing or
387
readout. For instance, the matrix effect is eliminated and the PCR inhibitors are avoided
388
which leads to a decrease of the background signals. The improvement of the washing
389
steps and the decrease of the time required for enrichment steps are also other
390
advantages. Finally, magnetic particles offer an attractive support to be incorporated in
391
magnetic actuated biosensors, microfluidic platforms or other devices.
392
Magnetic particles have also been successfully integrated in commercially available kits
393
for a rapid, simple and reliable detection of single pathogenic bacteria. However, there
394
are still only a few commercial kits available for simultaneous detection, with integrated
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395
magnetic nanoparticle functionality. As a conclusion the integration of magnetic
396
particles for the multiplex detection of bacteria is still in a preliminary stage, requiring
397
further studies due to their promising features.
398
Acknowledgments
400
Financial support from BioMaX “Novel diagnostic bioassays based on magnetic
401
particles”, Marie Curie Initial Training Networks (FP7-PEOPLE-2010-ITN) and the
402
Ministry of Economy and Competitiveness (MINECO), Madrid (Project BIO2013-
403
41242-R) are acknowledged.
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FIGURE CAPTIONS
406
Figure 1. Notifications for pathogens found in meat samples in 2012 and 2013.
407
Figure 2. Integration of magnetic particles in immunosensors and genosensors. After an
408
immunomagnetic separation step, (A) a magneto immunosensor and (B) a magneto
409
genosensor with electrochemical readout were reported for the detection of Salmonella
410
in milk. Adapted from [45].
411
Table 1. Strategies for the simultaneous detection of pathogenic bacteria based on
412
Nucleic acid amplification methods, Multiplex PCR based microfluidic systems and
413
DNA microarrays.
414
Table 2. Biosensors platforms based on the integration of nanomaterials for the
415
simultaneous detection of bacteria.
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Figure 3. Simultaneous detection of S. aureus, V. parahemolyticus, and S.
417
Typhimurium, using magnetic particles (MNPs) and multicolor upconversion
418
nanoparticles (UCNPs), as luminescence labels. UCNPs were modified with aptamers
419
(Apt1-3) specific for the different bacterium. Complementary oligonucleotides (c1-3DNA-
420
MPs) were immobilised on the MNPs. Reprinted with permission from [50]. Copyright
421
(2014) American Chemical Society.
422
Table 3. Commercial kits for the detection of pathogenic bacteria.
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Table 1 Preenrichment
Total assay time
LOD
Ref.
Agarose gel
artificially contaminated pork samples
overnight
~ 1h 40 min
9-670 CFU mL-1
[23]
Agarose gel
artificially contaminated meat samples
24 h
~ 1h 35 min
10–17 CFU g1 sample
[24]
>15 h
~ 1h 45 min
30h
1h 10 min
qPCR for Salmonella spp., E. coli, L. monocytogenes
Fluorescence
qPCR for Salmonella spp., E. coli, L. monocytogenes
Fluorescence
Nonaplex qPCR for L. monocytogenes, Campylobacter, Salmonella, enteropathogenic E. coli
Fluorescence
IMS-qPCR assay for Salmonella spp., Shigella spp., S. aureus
Fluorescence
IMS -mPCR assay for S. Typhimurium, E. coli, L. monocytogenes
Agarose gel
agarose gel electrophoresi s and GoldView™ agarose gel stained with GoldView™
Oscillatory-flow multiplex PCR PCR for S. enterica, E. coli, L. monocytogenes
Microchip capillary electrophoresis for V. parahemolyticus, Salmonella, E. coli, Shigella DNA microarray for Salmonella spp., Shigella spp., L. monocytogenes, E. coli
Capillary electrophoresis
Chemilumines -cence
DNA microarray for E. coli, S. enterica, L. monocytogenes, C. jejuni
Fluorescence
DNA microarray for 12 different bacterial strains using QDs
Fluorescence
[25]
1 CFU in 25 g sample
[52]
30h
55 min
5 CFU in 25 g sample
[53]
20 h
60 min
