Advances in Rapid Detection Methods for Foodborne Pathogens
Advances in Rapid Detection Methods for Foodborne Pathogens
Journal of Microbiology and Biotechnology. 2014. Mar, 24(3): 297-312
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : October 07, 2013
  • Accepted : December 22, 2013
  • Published : March 30, 2014
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About the Authors
Xihong Zhao
Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan
Chii-Wann Lin
Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan
Jun Wang
Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200701, Republic of Korea
Deog Hwan Oh
Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200701, Republic of Korea

Food safety is increasingly becoming an important public health issue, as foodborne diseases present a widespread and growing public health problem in both developed and developing countries. The rapid and precise monitoring and detection of foodborne pathogens are some of the most effective ways to control and prevent human foodborne infections. Traditional microbiological detection and identification methods for foodborne pathogens are well known to be time consuming and laborious as they are increasingly being perceived as insufficient to meet the demands of rapid food testing. Recently, various kinds of rapid detection, identification, and monitoring methods have been developed for foodborne pathogens, including nucleic-acid-based methods, immunological methods, and biosensor-based methods, etc . This article reviews the principles, characteristics, and applications of recent rapid detection methods for foodborne pathogens.
Foodborne pathogens are microorganisms ( i.e ., bacteria, viruses, and fungi) as well as a number of parasites, which are capable of infecting humans via contaminated food or water [24] . In particular, foodborne bacteria such as Escherichia coli O157:H7, Salmonella enterica , Staphylococcus aureus , Listeria monocytogenes , Campylobacter jejuni , Bacillus cereus , and other Shiga-toxin producing E. coli strains (non-O157 STEC), and Vibrio spp. are leading causes of foodborne diseases. In recent years, diseases caused by foodborne pathogens have become an important public health problem in the world, producing a significant rate of morbidity and mortality [72] . The global incidence of foodborne disease is difficult to estimate, but it has been reported that roughly 1 in 6 Americans in the United States (or 48 million people) gets sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases annually according to CDC 2011 Estimates [9] . A great proportion of these cases can be attributed to the contamination of food and drinking water. Additionally, diarrhea is a major cause of malnutrition in infants and young children [110] . Although there are 31 pathogens that have been identified as causing foodborne illnesses, Norovirus, Salmonella , Campylobacter , Staphylococcus aureus , Listeria monocytogenes , Clostridium perfringens , Toxoplasma gondii , and Escherichia coli O157:H7 have been generally found to be responsible for the vast majority of illnesses, hospitalizations, and deaths [9 , 100] .
The high prevalence of foodborne diseases in many developing countries suggests major underlying food safety problems; therefore, it is important to detect foodborne pathogens in order to reduce foodborne disease occurrence. Traditional methods for the detection of bacterial pathogens from foods depend on culturing the organisms on agar plates; it is a time-consuming process, taking 2-3 days for initial results, and up to more than 1 week for confirming the specific pathogenic microorganisms. It is obvious that culture and colony counting methods are inadequate. In order to prevent the spread of infectious diseases, ensure the food safety, and thereby to protect public health, there is an ever-increasing demand for more rapid methods of foodborne pathogen detection.
There is a wide variety of microorganisms that are able to produce toxins causing foodborne diseases, mainly S. aureus , Vibrio cholerae , Clostridium botulinum , C. perfringens , Bacillus cereus , and E. coli O157 [27] . Existing literatures report numerous methods developed for the detection of toxin-related genes and their toxin products. The detection methods of toxin-related genes are nucleic-acid-based methods, such as molecular amplification and hybridization probing. The detection methods of toxin products rely primarily on immunological assays, such as ELISA, lateral flow immunoassay and agglutination tests, and bioassays such as mouse neutralization testing and cytotoxicity assays in tissue culture, as well as biosensor-based assay [27 , 90] .
Recently, many researchers are focusing on the progress of rapid methods for foodborne pathogens. Novel molecular techniques for pathogens are being developed on various aspects of detection, such as sensitivity, rapidity, and selectivity, discrimination of the viable cell, and also suitability for in situ analysis. The immunological methods permit the rapid and sensitive analysis of a range of pathogens and toxins, especially with potential for on-site analysis. The emerging biosensor methods can detect foodborne pathogens in a much shorter time with sensitivity and selectivity comparable to the conventional methods and can potentially be used in the future as stand-alone devices for on-site monitoring. According to the main principle, these rapid detection methods can be classified into the following categories: nucleic-acid-based methods, immunological methods, and biosensor-based methods. The purpose of this paper is to review such rapid methods of detection and identification and to discuss some of the more recent and novel methods for the characterization of foodborne pathogens.
Nucleic-Acid-Based Methods
One of the advantages of nucleic-acid-based food pathogen detection assays is the high level of specificity, as they detect specific nucleic acid sequences in the target organism by hybridizing them to a short synthetic oligonucleotide complementary to the specific nucleic acid sequence. Several different types of nucleic-acid-based assays, including amplification, hybridization, microarrays, and biochips, have been developed for use as rapid methods to detect foodborne pathogens [92] .
- Simple PCR Method
PCR is the most well-known and established nucleic acid amplification technique for detecting pathogenic microorganisms [19] . In this method, double-stranded DNA is denatured into single strands, and specific primers or single-stranded (ss) oligonucleotides anneal to these DNA strands, followed by extension of the primers complementary to the singlestranded DNA, with a thermostable DNA polymerase. These steps are repeated, resulting in doubling of the initial number of target sequences with each cycle. This quantity of the products of amplification can be visualized as a band on an ethidium-bromide-stained electrophoresis gel. Identification based on PCR amplification of target genes by sequencing is considered to be a reliable technique when properly developed and validated for a certain species. With the distinct advantages of rapidity, specificity, sensitivity, and less samples over culture-based methods, many PCR assays for the detection and validation of foodborne bacteria and viruses in food have been developed and applied in food samples [38] .
PCR is also used for toxins detection by amplifying specific genes that encode bacterial toxins. PCR methods for toxin detection have been developed for a number of bacterial species, including V. cholera , B. cereus , E. coli , and S. aureus . In addition to PCR, a number of gene-specific hybridization probes have been designed and used for the detection of toxin genes in foodborne pathogens [77] .
- Multiplex PCR
Simultaneous amplification of more than one locus is required for a rapid detection of multiple microorganisms in a single reaction. It is a methodology referred to as multiplex PCR (mPCR), in which several specific primer sets are combined into a single PCR assay [10] . Apparently, the design of the primers is a key factor in the development of a multiplex PCR assay. There may be some interaction between the multiple primer sets, so the primer concentrations may have to be adjusted in order to generate reliable yields of all the PCR products. Meanwhile, the primer sets should be designed with a similar annealing temperature, while providing a method to distinguish between amplicons following thermal cycling. Today, mPCR can also be useful to define the structure of certain microbial communities and to evaluate community dynamics, such as during fermentation or in response to environmental variations. Kong et al . [46] described a rapid mPCR method allowing for the simultaneous detection of six commonly encountered waterborne pathogens in a single tube. The target genes used were the aerolysin ( aero ) gene of Aeromonas hydrophila , the invasion plasmid antigen H ( ipaH ) gene of Shigella flexneri , the attachment invasion locus ( ail ) gene of Yersinia enterocolitica , the invasion plasmid antigen B ( ipaB ) gene of Salmonella Typhimurium, the enterotoxin extracellular secretion protein ( epsM ) gene of Vibrio cholerae , and a species-specific region of the 16S-23S rDNA ( Vpara ) gene of Vibrio parahaemolyticus .
Park et al . [75] established a mPCR assay for the simultaneous detection of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus , and Listeria monocytogenes , in one tube. The mPCR employed the Escherichia coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B, and Listeria monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405, and 210 bp, respectively. Recently, Mukhopadhyay et al . [66] used fliCh7 and iap gene-specific primers to establish a multiplex-PCR assay for the simultaneous detection of Escherichia coli O157:H7 and Listeria monocytogenes . Meanwhile, they developed a modified method of enrichment and harvesting, leading to a highly sensitive and rapid single-reaction PCR detection of both pathogens.
- Quantitative PCR
Quantitative PCR (qPCR), also called real-time PCR, is an approach capable of continuously monitoring the PCR product formation throughout the reaction; it offers rapid, simultaneous amplification and sequence-specific-based detection of target genes and is increasingly being applied in food microbiology [41] . Using this method allows quantifying one specific microorganism in food and studying its behavior as a consequence of the influence of the environment ( i.e ., food composition, temperature, pH, oxygen, etc .) by studying expression of suitable target genes. Moreover, the real-time monitoring of the process means no need for post-amplification treatment of the samples, such as gel electrophoresis, reducing the time of analysis. Gomez et al . [33] developed a qPCR to quantify the total aerobic bacteria and fungi on fresh produce, using as reference the centrifugation water (CW) that comes up during processing instead of the food matrix itself. On average, 35% of the natural bacterial population and 64% of inoculated bacteria were recovered in the CW. Enumeration of cell number by qPCR did not differ significantly from plate assay and therefore, may replace it. This method could be an alternative to plate assays in order to get reliable information about the aerobic bacterial load of fresh-cut commodities in less than 5 h.
Derzelle et al . [22] developed a multiplex qPCR assay capable of detecting all known stx gene variants, including the highly divergent subtype stx 2f, and evaluated its performance in combination with two different internal amplification controls. The new screening method was tested with artificially and naturally contaminated food samples and compared with two stx -specific assays used routinely in their laboratory: a PCR-ELISA method and a real-time PCR system, which followed the recommendations from the International Organization for Standardization Technical Specification (ISO/TS) 13136 defining a method for the detection of the main pathogenic Shiga-toxin producing Escherichia coli (STEC) in foodstuffs. The results showed that the newly developed qPCR method performed equally as well as the PCR-ELISA test and the stx -IAC realtime PCR test when applied to the same 353 naturally contaminated test portions (99.7% concordance).
Fusco et al . [28] developed a TaqMan and a SYBR Green real time PCR assay for reliable identification and quantitative detection of S. aureus strains harboring the enterotoxin gene cluster, regardless of their variants. Using optimized qPCR conditions, the assay was able to quantitatively detect at least about 1 × 10 3 and 1 × 10 4 CFU of the pathogen per milliliter raw milk (10 and 100 CFU equivalents of egc + S. aureus per reaction mixture) by the SYBR Green and TaqMan qPCR assay, respectively.
Recently developed qPCR assays eliminated the post-PCR step by means of real-time monitoring of the PCR product generation, and the multiplex PCR approach has been implemented in the qPCR using a set of TaqMan probes labeled with different fluorescent dyes. Kim et al . [45] developed and proposed a multiplex qPCR assay for the simultaneous detection of V. cholerae , V. parahaemolyticus , and V. vulnificus , using zot , vmrA , and vuuA as target genes, respectively. The overall procedure took approximately 12 h, including the enrichment culture period; it yielded a method that was faster, simpler, and less costly than conventional culture-based methods. Using enrichment culture with alkaline peptone water and optimized ultiplex qPCR assay, they achieved a practical maximum sensitivity (10 0 CFU/g food homogenate) for each target species in all food matrices tested. Therefore, the method was shown to achieve a maximum sensitivity that meets the FDA guidelines (10 4 CFU/g) for acceptable levels of V. cholerae , V. parahaemolyticus , and V. vulnificus in seafood.
- Isothermal Amplification
Although PCR has been widely used in foodborne pathogens, it requires thermocycling to separate the double strands of DNA; this has limited its application in the lowresource settings. During the past two decades, many novel methods have been developed to amplify nucleic acids under isothermal conditions. These methods include loopmediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Isothermal amplification has simpler hardware requirements than PCR, as it does not require a thermal cycling system, and may even work with a simple water bath setup. Isothermal amplification techniques have better tolerance than PCR to some inhibitory materials that affect the molecular amplification efficiency [30] .
Loop-mediated isothermal amplification. Most recently, a novel nucleic acid amplification method known as LAMP has been demonstrated as a rapid, low-cost, easy operating, highly sensitive, and specific detection method applied in several fields [70] . This method relies on an autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment, which is different from PCR in that 4-6 primers are used to target 6-8 specific regions of the target gene. The amplification is performed under isothermal conditions between 59℃ and 65℃, and the amplicons are mixtures of many different sizes of stem loop DNAs with several inverted repeats of the target sequence and cauliflower-like structures with multiple loops. The reaction can be accelerated with additional one or two loop primers. LAMP reactions usually result in about 10 3 -fold or higher levels of amplification product with stem-loop DNAs in 60 min than conventional PCR. LAMP products can be observed with the naked eye by employing SYBR Green I dye instead of conventional gel electrophoresis analysis; the color of the solution changes to green in the presence of LAMP amplicons, whereas it remains orange for mixtures with no amplification.
The first foodborne pathogen application of the LAMP method was for the detection of stx A2 in Escherichia coli O157: H7 cells [62] . The mild permeabilization conditions and low isothermal temperatures used in the in situ LAMP method caused less cell damage than in situ PCR. The results showed that higher-contrast images were obtained with this method than with in situ PCR. Chen et al . [13] developed and evaluated a LAMP assay for identification and direct detection of acidophilic thermophilic bacteria (ATB) contaminants in pure juices. The LAMP method could detect 2.25 × 10 1 CFU/ml of ATB in juice samples within 2 h.
Recently, derivative LAMP assays, such as reversetranscription LAMP assay [12] , multiplex LAMP assay [40] , in situ LAMP assay [38 , 116] , and real-time reversetranscription LAMP assay [55] , have been developed and employed for the detection of various foodborne pathogens, such as Bacillus anthracis [79] , Vibrio parahaemolyticus [69 , 113 , 120] , Staphylococcus aureus [114] , Salmonella [13 , 116 , 119] , Pseudomonas aeruginosa [121] , Escherichia coli O157 [71 , 118] , and Listeria monocytogenes [105] .
Nucleic acid sequence-based amplification. NASBA is an isothermal amplification reaction for the detection of RNA or DNA, which was developed after PCR had begun gaining widespread attention [18] . The reaction typically consists of three enzymes, including T7 RNA polymerase, RNase H, and avian myeloblastosis virus (AMV) reverse transcriptase (RT), all of which act together to amplify sequences from an original single-stranded RNA template. The reaction also includes buffering agents and two specific primers and takes place at approximately 41℃ [35] .
NASBA is specific for target RNA or DNA sequences and has been gaining popularity owing to its wide range of applications for pathogen detection in clinical, environmental, and food samples [4] . Simpkins et al . [93] showed that NASBA can selectively amplify mRNA sequences from Salmonella enterica in a background of genomic DNA and demonstrated that NASBA could be a great means of assessing cell viability. Min and Baeumner [65] developed a NASBA assay for the detection of viable Escherichia coli . Baeumner et al . [5] confirmed the assay’s specificity for viable Escherichia coli by demonstrating that heat-killed cells did not produce a signal above the background of the instrumentation. Churruca et al . [17] developed a NASBA assay based on molecular beacons used for real-time detection of Campylobacter jejuni and Campylobacter coli in samples of chicken meat.
Real-time NASBA was proven to be the basis of sensitive and specific assays for detection, quantification, and analysis of RNA (and, in one case, DNA) targets [99] . Molecular beacons were used to generate fluorescence signals with NASBA assays for the detection of Vibrio cholerae [29] . More examples of the application of nucleic-acid-based techniques in food and other samples are presented in Table 1 .
Nucleic-acid-based techniques employed for pathogen detection.
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Immunological Methods
Immunological detection based on antigen-antibody bindings is widely used for determining foodborne pathogens. These assays rely mainly on the specific binding of an antibody to an antigen. A variety of antibodies have been employed in different assay types for the detection of foodborne pathogens and microbial toxins. The suitability of the antigen-antibody complex depends mainly on the antibodies’ specificity. In order to ensure the reliable detection of foodborne pathogens using antibody-based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first, as the physiological activities in cells are often altered in response to a stress [36] . Most polyclonal antibodies, derived from either rabbit or goat serum, contain a collection of antibodies with different cellular origins and, therefore, somewhat different specificities. Monoclonal antibodies are often more useful than polyclonal antibodies for specific detection of a molecule, since they provide an indefinite supply of a single antibody. With the development of monoclonal antibodies, immunological detection of microbial contamination has become more specific, sensitive, reproducible, and reliable, as many commercial immunological assays are available for the detection of a wide variety of microbes and their products [50] .
- Enzyme-Linked Immunosorbent Assay
One of the most widely used immunological assays for foodborne pathogens detection is enzyme-linked immunosorbent assay (ELISA), which is a very accurate and sensitive method for detecting antigens or haptens [101] . Traditional ELISA typically involves chromogenic reporters and substrates that produce some kind of observable color change to indicate the presence of antigen or analyte. The most powerful ELISA format is called the “sandwich” assay, because the antigen from the enrichment cultures to be measured is bound between two primary antibodies: the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust. The walls of wells in microtiter plates are the most commonly used solid support; however, ELISAs have also been designed using dipsticks, paddles, membranes, pipet tips, and other solid matrices [26] . Bolton et al . [6] described the BIOLINE Salmonella ELISA test for Salmonella spp., which was a rapid, easy, and convenient assay for the detection of Salmonella in foods and feeds. The limit of detection of the ELISA test kit was as low as 1 CFU/25 g sample with at least 4 of the 20 matrixes tested, and was found to be applicable to all sample types tested. The BIOLINE Salmonella ELISA test kit was granted AOAC-RI performance tested status.
Many foodborne toxins detection rely mainly on the presence of immunological reactions that are used to detect toxins. ELISA, the most commonly used in toxins detection, has been generated for staphylococcal enterotoxins A, B, C, and E and found to have detection levels of less than 0.5 μg/100 g in ground beef. ELISA has also been employed for the detection of botulinum toxins and enterotoxins produced by E. coli [27] .
- Lateral Flow Immunoassay
Although ELISA has been widely used in many laboratories, this method still requires various equipments and trained personnel. Therefore, rapid and cheap, yet still reliable methods that can be conducted and interpreted at the site of the contamination are needed. More and more on-site immunological techniques based on lateral flow immunoassays such as dipstick, immunochromatography, and immunofiltration are gaining attention in the area of pathogen, mycotoxin, and disease detection in the food industry and medicine [67] .
Lateral flow assays are a form of immunoassay in which the test sample flows along the solid substrate via capillary action. After the sample is applied to the test, it encounters a colored reagent (antibody or antigen labeled by colloidal latex or gold particles), which mixes with the sample and transits the substrate, encountering lines or zones that have been pretreated with an antibody or antigen. Depending on the analytes present in the sample, the colored reagent can become bound at the test line or zone [32] . Most lateral flow assays are basically designed to incorporate a visual response about 2-10 min after the application of the sample. Using these techniques allows simplifying the detection and minimizing the manipulations in order to provide accurate results with little or no instrumentation [25] .
Delmulle et al . [21] developed an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. The visual detection limit for aflatoxin B1 was 5 μg/kg. Jung et al . [42] developed a colloidal immunochromatographic strip for the detection of Escherichia coli O157:H7 in enriched samples, reporting that the minimum limit was 1.8 × 10 5 CFU/ml without enrichment and 1.8 CFU/ml after enrichment. Avidin and streptavidin are widely used in (strept)avidin-biotin system technology, which is based on their tight biotin-binding capability. These biotin-(strept)avidin-based methods enable both a signal amplification and a reduction in background activity, resulting in suitable analytical techniques to be used in many fields [84] .
The labels used in lateral flow immunoassay are mainly colloidal gold and monodisperse latex, labeled with colored, fluorescent, or magnetic tags. Dyed latexes and paramagnetic particles are available from a variety of sources, including Bangs Laboratories, Dynal, Merck/Estapor, and Magsphere. Magnetic, latex, metal, and semiconductor particles on the nanometer scale have unique optical, electronic, and structural properties that can be used in a variety of detection applications [78] .
With the development of semiqualitative and qualitative assays [2] as well as autoreading technologies, such as the Biosite Triage and Response Biomedical RAMP systems, Magnetic Assay Reader (MAR), Cozart’s DDS, or Rapiscan products such as American BioMedica Corporation’s Rapid Reader for their Rapid Screen, lateral flow immunoassay will be applied in ways that have the potential to create entirely new paradigms in high-sensitivity point-of-need testing on-site.
- Immunomagnetic Separation Assay
Immunomagnetic separation (IMS), a procedure that utilizes immunomagnetic beads (IMBs) as capturing reagents, has been developed for microbial isolation and identification. IMS is analogous to selective cultural enrichment, whereby the growth of other pathogen is suppressed while the target pathogen is allowed to grow. The separation process consists of two fundamental steps; first, the target cells are mixed with immunomagnetic particles for incubation of less than 1 h and separated by an appropriate magnetic separator; then, the magnetic complex is washed several times to remove the contaminants [60] . The use of IMS in assays is increasing because magnetic handling is fast, efficient, and only slightly affects the target analytes. Furthermore, various bioreactive molecules can be conjugated to the IMB surface for the immunoprecipitation, isolation, and identification of biomolecules (such as cells, pathogens, and proteins), or to improve the resolution of magnetic resonance imaging (MRI) [97] . Unlike the several days necessary to perform the conventional microbiological method and additional workup to elucidate the microbial status of any suspected colonies (more than 500 CFU in plate), DeCory et al . [20] developed and optimized a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic beadimmunoliposome (IMB/IL) fluorescence assay within a single 8 h work shift. The assay was able to identify samples containing Escherichia coli O157:H7 with 100% accuracy. The results highlighted the possible benefits of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of Escherichia coli O157:H7 in aqueous samples.
The growing importance of mass spectrometry for the identification and characterization of protein toxins produced by foodborne pathogens is a result of the improved sensitivity and specificity of mass-spectrometrybased techniques, especially when these techniques are combined with affinity methods. Schlosser et al . [87] reported a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). In this method, an affinity molecular probe was prepared by immobilizing the anti-SEB antibody on the surface of paratoluene-sulfonyl-functionalized monodisperse magnetic particles and was used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of the magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution.
Other technologies relying on the Ab-Ag binding mechanism have also been developed and applied in detection of foodborne pathogens and toxins. Lee and Deininger [49] used IMS and ATP bioluminescence for the selective capture of target bacteria and their quantification, respectively. The method consisted of trapping bacteria on a filter, resuspending them in a small amount of buffer, and washing the suspension with an antibody-coated magnetic bead mixture specific to the bacterial species of interest. A detection limit of about 20 CFU/100 ml was achieved, which was well below the action limits of 300 CFU/ml (daily event), or a 30 day moving average of 126 CFU/100 ml set by US EPA. The entire procedure took less than 1 h to perform without an enrichment step. The study demonstrated that the system combining IMS with ATP bioluminescence was effective and expedient for detecting Escherichia coli in beach water.
Biosensor-Based Methods
Biosensors have recently been defined as analytical devices incorporating a biological material ( e.g ., tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, natural products, etc .), a biologically derived material ( e.g ., recombinant antibodies, engineered proteins, aptamers, etc .), or a biomimic ( e.g ., synthetic catalysts, combinatorial ligands, and imprinted polymers) intimately associated with or integrated within a physicochemical transducer or transducing microsystem, which may be optical, electrochemical, thermometric, piezoelectric, magnetic, or micromechanical [48] . Biosensors are devices for pathogen detection and generally consist of at least three elements, including a biological capture molecule ( e.g ., probes or antibodies), a method of converting capture molecule-target interactions into a signal, and a data output system [48 , 100] . The greatest advantageous aspects of biosensors are those that enable fast or real-time detection, portability, and multi-pathogen detection for both field and laboratory analyses. The advantages of fast or real-time detection can provide almost immediate interactive information on the food materials, which enable users to take corrective measures before consumption or further contamination occurs [82] . It has been reported that biosensors have been developed and applied to the microbial analysis of foodborne pathogens, including Escherichia coli O157:H7, Staphylococcus aureus , Salmonella , and Listeria monocytogenes , as well as various microbial toxins such as staphylococcal enterotoxins and mycotoxins [3] . Different modes of biosensor-based foodborne pathogen detection are given in Table 2 .
Different modes of biosensor-based foodborne pathogen detection.
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Different modes of biosensor-based foodborne pathogen detection.
- Optical Biosensors
Optical biosensors are a powerful alternative to conventional analytical techniques, due to their particularly high specification and sensitivity, as well as their small size and cost-effectiveness [58] . Biosensor detection typically relies on an enzyme system, which catalytically converts analytes into products that can be oxidized or reduced at a working electrode and maintained at a specific potential. One of the best advantages of this optical transducer is the low cost and the use of biodegradable electrodes. An optical biosensor is a compact analytical device containing a biological sensing element integrated or connected to an optical transducer system [23] . Optical biosensor technology can be classified into several subclasses based on absorption, reflection, refraction, Raman, infrared, chemiluminescence, dispersion, fluorescence, and phosphorescence. In the past decade, various kinds of optical biosensors for the rapid detection of pathogens, toxins, and contaminants in the food industry have been developed [100] . The main advantage of this technique is the real-time binding reaction detection, allowing kinetic evaluation of affinity interactions and, in addition, the low cost of the instrumentation required. Optical biosensors require a suitable spectrometer to record the spectral chemical properties of the analyte. A common method that employs the techniques of optical detection using reflectance spectroscopy for detection of foodborne pathogens is surface plasmon resonance (SPR).
SPR is a collective oscillation of free charges (conduction electrons) present at the interface of two media (metal-dielectric) with permittivities of opposite sign [1] . Receptors or antibodies initially immobilized on the surface of a thin film of precious metal, deposited on the reflecting surface of an optically transparent waveguide, are used to capture the various target pathogens. The sensing surface is located above or below a high-index resonant layer and a lowindex coupling layer. When a visible or near-infrared radiation (IR) is passed through the waveguide in the correct manner, the interaction of light with the electron cloud in the metal generates a strong resonance. Binding of the pathogen to the metal surface causes a shift in resonance to longer wavelengths, and the corresponding amount of shift reflects the concentration of bound pathogens [3] . The main drawbacks of current SPR technique lay in its complexity (specialized staff is required), high cost of equipment, and the large size of most currently available instruments. For this reason, the miniature SPR instrument and disposable cartridge and biochip were developed for real-time genetic detection in a cost-effective manner. This system included a disposable SPRLAMP cartridge made of PMMA with a PC prism and a simple SPR imaging system with temperature control for LAMP amplification [16] .
Wang et al . [107] developed a SPR immunosensor for the detection of Escherichia coli O157:H7 by means of a new subtractive inhibition assay. Their results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 10 4 to 3.0 × 10 8 CFU/ml, where the limit of detection was 3.0 × 10 4 CFU/ml. The limit of detection subtractive inhibition assay method was reduced by one order of magnitude, compared with direct SPR by immobilizing antibodies on the chip and ELISA for E. coli O157:H7 (limit of detection: both 3.0 × 10 5 CFU/ml).
Several commercial instruments using SPR techniques are available from companies such as BIAcore and Biosensing Instruments Inc. Commercial SPR instruments have a detection limit of 10 5 CFU/ml for Listeria monocytogenes . The commercially available low-cost SPREETA SPR biosensor was reported to detect an E. coli O157:H7 enterotoxin, stx 1, with a detection limit of 300 pmol compared with a bulk acoustic wave sensor [96] .
- Piezoelectric Biosensors
Piezoelectric biosensors, which are capable of sensitive detection of minute amounts of analytes according to a linear relationship between the deposited mass and its frequency response, are an effective alternative to established label-free optical sensors, such as surface plasmon resonance spectroscopy and interferometry [11] . Piezoelectric biosensors have been widely used, and their performance for studies of affine interactions was extensively referred.
Olfactory sensing of specific volatile organic compounds released by the bacterial pathogens is one of the more outstanding ways to determine contamination in food products. Sankaran et al . [86] used a computational simulation to determine the biomimetic peptide-based sensing material to be deposited on the quartz crystal microbalance (QCM) sensor for detecting specific gases (alcohols) at low concentrations in food samples. The results showed that the developed QCM sensors were sensitive to 1-hexanol as well as 1-pentanol as predicted by the simulation algorithm. The estimated lower detection limits of the QCM sensors for detecting 1-hexanol and 1-pentanol were 2-3 ppm and 3-5 ppm, respectively. This report demonstrated the applicability of a simulation-based peptide sequence that mimics the olfactory receptor for sensing specific gases.
Salmain et al . [85] successfully designed a direct, labelfree immunosensor for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions, using the quartz crystal microbalance with dissipation (QCM-D) as a transduction method. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1,000 or 2,000 ng/ml, with a detection limit of 20 ng/ml. The total time for analysis was 15 min. The study indicated that such systems had a considerable amount of potential for the rapid and reliable detection of targets at trace amounts of pathogens in various environments.
- Immunosensors
Immunosensors, which are based on specific antibodyantigen interactions, detect antigen binding to antibodies by immobilizing the reaction to the surface of a transducer, which converts surface change parameters into a detectable electric signal [32] . It is difficult to measure immunological reactions in real time owing to the diffusion limitations of antigens to immobilized antibodies, particularly for low levels of contaminants. However, most immunosensors produce results within 20-90 min, which is close to real time compared with conventional techniques and classical ELISAs. Moreover, the results of immunosensors are read via digital signals and are not as dependant on personal factors such as bias, fatigue, level of training, or visual disorders. However, this property is also shared by microtiter plate spectrophotometric immunoassays [98] . Chen [15] reported a new conductometric immunebiosensor for the detection of staphylococcal enterotoxin B (SEB) based on immobilization of horseradish peroxidase (HRP)-labeled SEB antibody (HRP-anti-SEB) onto a nanogold/ chitosan-multiwalled carbon nanotube (Au/CTS-MWNT)-functionalized biorecognition interface. The results showed that under optimal conditions, the proposed immunebiosensor exhibited a good conductometric response relative to SEB concentration in a linear range from 0.5 to 83.5 ng/ml, with a correlation coefficient of 0.998.
- Electrochemical Biosensors
Electrochemical-based detection methods are further transduction-based systems that have been used for identifying and quantifying foodborne pathogens. Electrochemical biosensors can be classified into amperometric, potentiometric, impedimetric, and conductometric responses, based on observed parameters such as current, potential, impedance, and conductance, respectively [100] . Electrochemical biosensors developed for the simultaneous multiplexed analysis of foodborne pathogens primarily use electrochemical impedance spectroscopy as the transduction technique, thus providing label-free, on-line, high-throughput devices for bacterial detection [76] . Impedance spectroscopy is a powerful method for the study of conducting materials and interfaces. Through this technique, a cyclic function of small amplitude and variable frequency is applied to a transducer, and the resulting current is used to calculate the impedance at each of the probed frequencies [48] . Impedance biosensors for the detection of foodborne pathogens are based on the measurement of changes in the electrical properties of bacterial cells when they are attached to, or associated with, the electrodes [115] . Furthermore, the advantages in microfabrication techniques have enabled the use of microfabricated microarray electrodes for impedance detection, and the miniaturization of impedance microbiology into a chip assay.
Louie et al . [57] developed an impedance-based, fieldable biosensor system to detect Escherichia coli O157:H7 and Salmonella spp. The portable biosensor system used a variety of disposable analyte-specific sensor modules, each of which could be used to quantitatively determine specific analytes. The response for each sensor was rapid, and stable readings could be obtained in less than 1 min. Despite the development of a portable reagentless impedance biosensor that allows rapid detection of specific foodborne pathogens, however, no real foodborne or clinical sample application was considered.
There seems to be a lot of interest in the development of integrated biosensors for the detection of multiple biologically relevant species. A miniaturized biosensor device composed of a probe, sampler, detector, amplifier, and logic circuitry for monitoring infectious pathogens is an attractive alternative to existing instrumentation. Normal biosensors and biochips employ only one type of bioreceptor as probes ( i.e ., either nucleic acid, enzyme, or antibody probes). The multifunctional biochip (MFB) is an integrated multi-array biochip, designed by combining integrated circuit elements, an electro-optics excitation/detection system, and bioreceptor probes into a self-contained and integrated microdevice [103] . The MFB is a superior system that can detect multiple specific analytes simultaneously and offer information on both gene mutation (with DNA probes) and protein expression (with antibody probes) simultaneously. Vo-Dinh et al . [103] described a MFB, which used two different types of bioreceptors, including nucleic acid and antibody probes, on a single platform. The multifunctional capability of the MFB device for biomedical diagnostics was illustrated by the measurements of DNA probes specific to gene fragments of Bacillus anthracis and antibody probes targeted to Escherichia coli . The results showed that the calibration curves for monitoring pathogenic species illustrated the capability of the device for medical diagnostics and for quantitative detection of pathogenic agents.
Cao et al . [8] described a rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on a microchip. They employed a biotin-modified DNA, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP), in order to obtain the HRPlabeled DNA probe, and hybridized it with its complementary target. After hybridization, the mixture containing dsDNAHRP, excess ssDNA-HRP, and remaining avidin-HRP was separated. With this protocol, the limits of quantification for the hybridization assay of 21- and 39-mer DNA fragments were 8 × 10 -12 M and 1.2 × 10 -11 M, respectively. The method was applied satisfactorily in the analysis of Escherichia coli genomic DNA.
Wang et al . [104] described a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combining nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic beadbased microfluidic system. LAMP amplification of the target genes was performed via the incorporation of a built-in micro temperature control module, followed by spectrophotometric analysis of the optical density of the LAMP amplicons. The results showed that the limit of detection for MRSA in clinical samples was approximately 10 fg/ml by performing this diagnostic assay in the magnetic bead-based microfluidic system within 60 min.
Future Perspectives
Traditional foodborne pathogen detection methods, although sensitive enough, are often too time-consuming for practical use, taking days to a week to perform. Therefore, new methods that overcome this performance limitation are required. Recently, several methods have been explored and developed for the rapid detection of foodborne pathogens. However, most of them still require improvement in sensitivity, selectivity, or accuracy to be of any practical use.
Nucleic-acid-based methods have high sensitivity and require a shorter time than conventional culture-based techniques for detection of foodborne pathogens and toxins, but most of them require trained personnel and expensive instruments, which limit their use in a practical environment. The emerging isothermal amplification methods such as LAMP and NASBA may have a good prospect for detection of pathogens and toxins in resourcelimit settings. The development of nucleic-acid-based methods and immunological methods helped improve the time required to yield results. The specificity and the sensitivity of immunological methods depend on the binding strength of the specific antibody to its antigen, and they work well for food matrixes without interfering factors such as other non-target cells, DNA, and proteins. Biosensors-based methods are easy to perform without training, and yield results in real-time detection of foodborne pathogens and toxins with high sensitivity and selectivity comparable to the culture-based methods. However, they still need to be improved in food matrixes detection.
All assays available for food diagnostics require some degree of sample preparation, which is a very important factor for rapid and conventional detection methods, and also a bottleneck for the advanced rapid methods. More studies regarding the separation techniques of microorganisms from the food matrix are required, as well as for sample concentration prior to detection by immunological, nucleicacid-based, or biosensor assays. Preconcentration is the preferred choice, as it can enhance sensitivity several folds by increasing the number of target organisms per unit volume at a relatively low cost. Several available modes of preconcentration are used, including filtration, sizefractionation, centrifugation, and immunomagnetic separation, or combinations of these methods.
The possibilities of combining various rapid methods, including nucleic-acid-based methods, immunologicalbased methods, and biosensor-based methods should be further exploited. With the correct application of a number of these technologies simultaneously, broader ranging and more accurate technologies could be developed. Antibodies can be modified to capture specific cells, which may then be detected by a nucleic-acid-based method. Various nucleic acid amplified products can be quantified using immunoassays. The trend in immunoassays and nucleic-acid-based methods should result in the quantitative detection of microorganisms and the simultaneous determination of more than one pathogen or toxin. For immunological-based methods, further study of the application of biosensor chips may result in multiplex analyte assays. Biosensors must prove that they are capable of reaching at least the same detection levels as traditional methods (between 10 and 100 CFU/ml) in order to strengthen their appeal in food microbiology applications, not to mention the costeffectiveness and time efficiency. Despite the numerous research efforts made during the past decades and in recent years for foodborne pathogen detection, current technologies still entail room for improvement. Since foodborne pathogens are mostly present in very low numbers (<100 CFU/g) and in the presence of millions of other bacteria, they are not easily detected. Therefore, a detection method that is reliable, accurate, rapid, simple, sensitive, selective, and cost-effective would be ideal. Such methods of pathogen detection would offer a great commercial advantage in the food industry and related fields. Moreover, the trend of crossing various methods will generate novel devices or methodologies to strengthen the advantages of rapid detection methods.
In summary, there are a host of promising applications in the field of rapid and automated detection methods for foodborne pathogens. Given the broad applicability and the great potential of such methods, there is still a great chance for further developments in the near future.
Abbas A , Linman MJ , Cheng QA 2011 New trends in instrumental design for surface plasmon resonance-based biosensors. Biosens. Bioelectron. 26 1815 - 1824    DOI : 10.1016/j.bios.2010.09.030
Arora P , Sindhu A , Dilbaghi N , Chaudhury A 2011 Biosensors as innovative tools for the detection of food borne pathogens. Biosens. Bioelectron. 28 1 - 12    DOI : 10.1016/j.bios.2011.06.002
Asiello PJ , Baeumner AJ 2011 Miniaturized isothermal nucleic acid amplification, a review. Lab. Chip 11 1420 - 1430    DOI : 10.1039/c0lc00666a
Baeumner AJ , Cohen RN , Miksic V , Min J 2003 RNA biosensor for the rapid detection of viable Escherichia coli in drinking water. Biosens. Bioelectron. 18 405 - 413    DOI : 10.1016/S0956-5663(02)00162-8
Bolton FJ , Fritz E , Poynton S , Jensen T 2000 Rapid enzyme-linked immunoassay for detection of Salmonella in food and feed products: performance testing program. J. AOAC Int. 83 299 - 303
Campbell GA , Mutharasan R 2006 Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors detect Bacillus anthracis at 300 spores/ml. Biosens. Bioelectron. 21 1684 - 1692    DOI : 10.1016/j.bios.2005.08.001
Cao W , Su M , Zhang S 2010 Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip. Electrophoresis 31 659 - 665    DOI : 10.1002/elps.200900538
CDC 2011 CDC Estimates of Foodborne Illness in the United States. [Online.]
Chamberlain JS , Gibbs RA , Ranier JE , Nguyen PN , Caskey CT 1988 Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16 11141 - 11156    DOI : 10.1093/nar/16.23.11141
Chen HM , Lin CW 2007 Hydrogel-coated streptavidin piezoelectric biosensors and applications to selective detection of Strep-Tag displaying cells. Biotechnol. Prog. 23 741 - 748    DOI : 10.1021/bp060328h
Chen HT , Zhang J , Sun DH , Ma LN , Liu XT , Cai XP , Liu YS 2008 Development of reverse transcription loopmediated isothermal amplification for rapid detection of H9 avian influenza virus. J. Virol. Methods 151 200 - 203    DOI : 10.1016/j.jviromet.2008.05.009
Chen J , Ma XY , Yuan YW , Zhang W 2011 Sensitive and rapid detection of Alicyclobacillus acidoterrestris u sing loop-mediated isothermal amplification. J. Sci. Food Agric. 91 1070 - 1074    DOI : 10.1002/jsfa.4285
Chen SY , Wang F , Beaulieu JC , Stein RE , Ge BL 2011 Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification. Appl. Environ. Microbiol. 77 4008 - 4016    DOI : 10.1128/AEM.00354-11
Chen ZG 2008 Conductometric immunosensors for the detection of staphylococcal enterotoxin B based bioelectrocalytic reaction on micro-comb electrodes. Bioproc. Biosyst. Eng. 31 345 - 350    DOI : 10.1007/s00449-007-0168-2
Chuang TL , Wei SC , Lee SY , Lin CW 2012 A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification. Biosens. Bioelectron. 32 89 - 95    DOI : 10.1016/j.bios.2011.11.037
Churruca E , Girbau C , Martinez I , Mateo E , Alonso R , Fernandez-Astorga A 2007 Detection of Campylobacter jejuni and Campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons. Int. J. Food Microbiol. 117 85 - 90    DOI : 10.1016/j.ijfoodmicro.2007.02.007
Compton J 1991 Nucleic acid sequence-based amplification. Nature 350 91 - 92    DOI : 10.1038/350091a0
de Boer E , Beumer RR 1999 Methodology for detection and typing of foodborne microorganisms. Int. J. Food Microbiol. 50 119 - 130    DOI : 10.1016/S0168-1605(99)00081-1
DeCory TR , Durst RA , Zimmerman SJ , Garringer LA , Paluca G , DeCory HH , Montagna RA 2005 Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method. Appl. Environ. Microbiol. 71 1856 - 1864    DOI : 10.1128/AEM.71.4.1856-1864.2005
Delmulle BS , De Saeger SM , Sibanda L , Barna-Vetro I , Van Peteghem CH 2005 Development of an immunoassaybased lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. J. Agric. Food Chem. 53 3364 - 3368    DOI : 10.1021/jf0404804
Derzelle S , Grine A , Madic J , de Garam CP , Vingadassalon N , Dilasser F 2011 A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products. Int. J. Food Microbiol. 151 44 - 51    DOI : 10.1016/j.ijfoodmicro.2011.07.039
Dey D , Goswami T 2011 Optical Biosensors: A revolution towards quantum nanoscale electronics device fabrication. J. Biomed. Biotechnol. 2011 348218 -    DOI : 10.1155/2011/348218
Dongyou L 2010 Molecular Detection of Foodborne Pathogens. CRC Press Boca Raton.
Dwivedi HP , Jaykus LA 2011 Detection of pathogens in foods: the current state-of-the-art and future directions. Crit. Rev. Microbiol. 37 40 - 63    DOI : 10.3109/1040841X.2010.506430
Feng P 1997 Impact of molecular biology on the detection of foodborne pathogens. Mol. Biotechnol. 7 267 - 278    DOI : 10.1007/BF02740817
Foley SL , Grant K 2007 Molecular techniques of detection and discrimination of foodborne pathogens and their toxins, p. 485-510. In Simjee S (ed.). Infectious Disease: Foodborne Diseases. Humana Press Inc Totowa, NJ.
Fusco V , Quero GM , Morea M , Blaiotta G , Visconti A 2011 Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR. Int. J. Food Microbiol. 144 528 - 537    DOI : 10.1016/j.ijfoodmicro.2010.11.016
Fykse EM , Skogan G , Davies W , Olsen JS , Blatny JM 2007 Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 73 1457 - 1466    DOI : 10.1128/AEM.01635-06
Gill P , Ghaemi A 2008 Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids 27 224 - 243    DOI : 10.1080/15257770701845204
Gill P , Ramezani R , Amiri MVP , Ghaemi A , Hashempour T , Eshraghi N 2006 Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M-tuberculosis. Biochem. Biophys. Res. Commun. 347 1151 - 1157    DOI : 10.1016/j.bbrc.2006.07.039
Gizeli E , Lowe CR 1996 Immunosensors. Curr. Opin. Biotechnol. 7 66 - 71    DOI : 10.1016/S0958-1669(96)80097-8
Gomez P , Pagnon M , Egea-Cortines M , Artes F , Weiss J 2010 A fast molecular nondestructive protocol for evaluating aerobic bacterial load on fresh-cut lettuce. Food Sci. Technol. Int. 16 409 - 415    DOI : 10.1177/1082013210366882
Grothaus GD , Bandla M , Currier T , Giroux R , Jenkins GR , Lipp M 2006 Immunoassay as an analytical tool in agricultural biotechnology. J. AOAC Int. 89 913 - 928
Guatelli JC , Whitfield KM , Kwoh DY , Barringer KJ , Richman DD , Gingeras TR 1990 Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87 7797 -    DOI : 10.1073/pnas.87.19.7797-a
Hahm BK , Bhunia AK 2006 Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes. J. Appl. Microbiol. 100 1017 - 1027    DOI : 10.1111/j.1365-2672.2006.02814.x
Hahn MA , Keng PC , Krauss TD 2008 Flow cytometric analysis to detect pathogens in bacterial cell mixtures using semiconductor quantum dots. Anal. Chem. 80 864 - 872    DOI : 10.1021/ac7018365
Hill WE 1996 The polymerase chain reaction: applications for the detection of foodborne pathogens. Crit. Rev. Food Sci. Nutr. 36 123 - 173    DOI : 10.1080/10408399609527721
Ikeda S , Takabe K , Inagaki M , Funakoshi N , Suzuki K 2007 Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol. Int. 57 594 - 599    DOI : 10.1111/j.1440-1827.2007.02144.x
Iseki H , Alhassan A , Ohta N , Thekisoe OMM , Yokoyama N , Inoue N 2007 Development of a multiplex loopmediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia p arasites. J. Microbiol. Methods 71 281 - 287    DOI : 10.1016/j.mimet.2007.09.019
Jasson V , Jacxsens L , Luning P , Rajkovic A , Uyttendaele M 2010 Alternative microbial methods: an overview and selection criteria. Food Microbiol. 27 710 - 730    DOI : 10.1016/
Jung BY , Jung SC , Kweon CH 2005 Develop ment of a rapid immunochromatographic strip for detection of Escherichia coli O157. J. Food Prot. 68 2140 - 2143
Kawasaki S , Fratamico PM , Horikoshi N , Okada Y , Takeshita K , Sameshima T , Kawamoto S 2009 Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing. Foodborne Pathog. Dis. 6 81 - 89    DOI : 10.1089/fpd.2008.0153
Kawasaki S , Horikoshi N , Okada Y , Takeshita K , Sameshima T , Kawamoto S 2005 Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. J. Food Prot. 68 551 - 556
Kim HJ , Lee HJ , Lee KH , Cho JC 2012 Simultaneous detection of pathogenic Vibrio species using multiplex realtime PCR. Food Control 23 491 - 498    DOI : 10.1016/j.foodcont.2011.08.019
Kong RY , Lee SK , Law TW , Law SH , Wu RS 2002 Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR. Water Res. 36 2802 - 2812    DOI : 10.1016/S0043-1354(01)00503-6
Lan YB , Wang SZ , Yin YG , Hoffmann WC , Zheng XZ 2008 Using a surface plasmon resonance biosensor for rapid detection of Salmonella Typhimurium in chicken carcass. J. Bionic Eng. 5 239 - 246    DOI : 10.1016/S1672-6529(08)60030-X
Laura A , Gilda D , Claudio B , Cristina G , Gianfranco G 2011 A lateral flow immunoassay for measuring ochratoxin A: development of a single system for maize, wheat and durum wheat. Food Control 22 1965 - 1970    DOI : 10.1016/j.foodcont.2011.05.012
Lazcka O , Del Campo FJ , Munoz FX 2007 Pathogen detection: A perspective of traditional methods and biosensors. Biosens. Bioelectron. 22 1205 - 1217    DOI : 10.1016/j.bios.2006.06.036
Lee JY , Deininger RA 2004 Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence. Luminescence 19 31 - 36    DOI : 10.1002/bio.753
Leonard P , Hearty S , Brennan J , Dunne L , Quinn J , Chakraborty T , O’Kennedy R 2003 Advances in biosensors for detection of pathogens in food and water. Enzyme Microb. Technol. 32 3 - 13    DOI : 10.1016/S0141-0229(02)00232-6
Li Y , Cheng P , Gong JH , Fang LC , Deng J , Liang WB , Zheng JS 2012 Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens. Anal. Biochem. 421 227 - 233    DOI : 10.1016/j.ab.2011.10.049
Li Y , Mustapha A 2004 Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR. J. Food Prot. 67 27 - 33
Lin WS , Cheng CM , Van KT 2010 A quantitative PCR assay for rapid detection of Shigella species in fresh produce. J. Food Prot. 73 221 - 233
Lin YH , Chen SH , Chuang YC , Lu YC , Shen TY , Chang CA , Lin CS 2008 Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screenprinted carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7. Biosens Bioelectron. 23 1832 - 1837    DOI : 10.1016/j.bios.2008.02.030
Liu Y , Chuang CK , Chen WJ 2009 In situ reversetranscription loop-mediated isothermal amplification (in situ RT-LAMP) for detection of Japanese encephalitis viral RNA in host cells. J. Clin. Virol. 46 49 - 54    DOI : 10.1016/j.jcv.2009.06.010
Loens K , Beck T , Goossens H , Ursi D , Overdijk M , Sillekens P , Ieven M 2006 Development of conventional and real-time nucleic acid sequence-based amplification assays for detection of Chlamydophila pneumoniae in respiratory specimens. J. Clin. Microbiol. 44 1241 - 1244    DOI : 10.1128/JCM.44.4.1241-1244.2006
Louie AS , Marenchic IG , Whelan RH 1998 A fieldable modular biosensor for use in detection of foodborne pathogens. Field Anal. Chem. Technol. 2 371 - 377    DOI : 10.1002/(SICI)1520-6521(1998)2:6<371::AID-FACT7>3.0.CO;2-F
Luo XL , Xu JJ , Zhao W , Chen HY 2004 Glucose biosensor based on ENFET doped with SiO2 nanoparticles. Sensors Actuat B Chem. 97 249 - 255    DOI : 10.1016/j.snb.2003.08.024
Magliulo M , Simoni P , Guardigli M , Michelini E , Luciani M , Lelli R , Roda A 2007 A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria. J. Agric. Food Chem. 55 4933 - 4939    DOI : 10.1021/jf063600b
Mandal PK , Biswas AK , Choi K , Pal U 2011 Methods for rapid detection of foodborne pathogens: an overview. Am. J. Food Technol. 6 87 - 102    DOI : 10.3923/ajft.2011.87.102
Martinon A , Wilkinson MG 2011 Selection of optimal primer sets for use in a duplex sybr green-based, real-time polymerase chain reaction protocol for the detection of listeria monocytogenes and staphyloccocus aureus in foods. J. Food Saf. 31 297 - 312    DOI : 10.1111/j.1745-4565.2011.00301.x
Maruyama F , Kenzaka T , Yamaguchi N , Tani K , Nasu M 2003 Detection of bacteria carrying the stx2 gene by in situ loop-mediated isothermal amplification. Appl. Environ. Microbiol. 69 5023 - 5028    DOI : 10.1128/AEM.69.8.5023-5028.2003
McEgan R , Fu TJ , Warriner K 2009 Concentration and detection of Salmonella in mung bean sprout spent irrigation water by use of tangential flow filtration coupled with an amperometric flowthrough enzyme-linked immunosorbent assay. J. Food Prot. 72 591 - 600
Meeusen CA , Alocilja EC , Osburn WN 2005 Detection of E. coli O157:H7 using a miniaturized surface plasmon resonance biosensor. Trans. ASAE 48 2409 - 2416    DOI : 10.13031/2013.20067
Min J , Baeumner AJ 2002 Highly sensitive and specific detection of viable Escherichia coli in drinking water. Anal. Biochem. 303 186 - 193    DOI : 10.1006/abio.2002.5593
Mukhopadhyay A , Mukhopadhyay UK 2007 Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli O157:H7 and Listeria monocytogenes. J. Microbiol. Methods 68 193 - 200    DOI : 10.1016/j.mimet.2006.07.009
Muldoon MT , Teaney G , Li J , Onisk DV , Stave JW 2007 Bacteriophage-based enrichment coupled to immunochromatographic strip-based detection for the determination of Salmonella in meat and poultry. J. Food Prot. 70 2235 - 2242
Nam HM , Srinivasan V , Gillespie BE , Murinda SE , Oliver SP 2005 Application of SYBR green real-time PCR assay for specific detection of Salmonella spp .in dairy farm environmental samples. Int. J. Food Microbiol. 102 161 - 171    DOI : 10.1016/j.ijfoodmicro.2004.12.020
Nemoto J , Ikedo M , Kojima T , Momoda T , Konuma H , Hara-Kudo Y 2011 Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus. J. Food Prot. 74 1462 - 1467    DOI : 10.4315/0362-028X.JFP-10-519
Notomi T , Okayama H , Masubuchi H , Yonekawa T , Watanabe K , Amino N , Hase T 2000 Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28 E63 -    DOI : 10.1093/nar/28.12.e63
Ohtsuka K , Tanaka M , Ohtsuka T , Takatori K , Hara-Kudo Y 2010 Comparison of detection methods for Escherichia coli O157 in beef livers and carcasses. Foodborne Pathog. Dis. 7 1563 - 1567    DOI : 10.1089/fpd.2010.0585
Oliver SP , Jayarao BM , Almeida RA 2005 Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. Foodborne Pathog. Dis. 2 115 - 129    DOI : 10.1089/fpd.2005.2.115
Ong KG , Zeng KF , Yang XP , Shankar K , Ruan CM , Grimes CA 2006 Quantification of multiple bioagents with wireless, remote-query magnetoelastic microsensors. IEEE Sensors J. 6 514 - 523    DOI : 10.1109/JSEN.2006.874450
Pal S , Ying W , Alocilja EC , Downes FP 2008 Sensitivity and specificity performance of a direct-charge transfer biosensor for detecting Bacillus cereus in selected food matrices Biosys Eng. 99 461 - 468    DOI : 10.1016/j.biosystemseng.2007.11.015
Park YS , Lee SR , Kim YG 2006 Detection of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in kimchi by multiplex polymerase chain reaction (mPCR). J. Microbiol. 44 92 - 97
Pedrero M , Campuzano S , Pingarron JM 2009 Electroanalytical sensors and devices for multiplexed detection of foodborne pathogen microorganisms. Sensors 9 5503 - 5520    DOI : 10.3390/s90705503
Planche T , Aghaizu A , Holliman R , Riley P , Poloniecki J , Breathnach A , Krishna S 2008 Diagnosis of Clostridium difficile infection by toxin detection kits: a systematic review. Lancet Infect. Dis. 8 777 - 784    DOI : 10.1016/S1473-3099(08)70233-0
Posthuma-Trumpie GA , Korf J , van Amerongen A 2009 Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 393 569 - 582    DOI : 10.1007/s00216-008-2287-2
Qiao YM , Guo YC , Zhang XE , Zhou YF , Zhang ZP , Wei HP 2007 Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol. Lett. 29 1939 - 1946    DOI : 10.1007/s10529-007-9472-9
Rahman S , Lipert RJ , Porter MD 2006 Rapid screening of pathogenic bacteria using solid phase concentration and diffuse reflectance spectroscopy. Anal. Chim. Acta 569 83 - 90    DOI : 10.1016/j.aca.2006.03.083
Rao VK , Rai GP , Agarwal GS , Suresh S 2005 Amperometric immunosensor for detection of antibodies of Salmonella Typhi in patient serum. Anal. Chim. Acta 531 173 - 177    DOI : 10.1016/j.aca.2004.10.015
Rasooly A , Herold KE 2006 Biosensors for the analysis of food- and waterborne pathogens and their toxins. J. AOAC Int. 89 873 - 883
Rodriguez-Lazaro D , Jofre A , Aymerich T , Hugas M , Pla M 2004 Rapid quantitative detection of Listeria monocytogenes in meat products by real-time PCR. Appl. Environ. Microbiol. 70 6299 - 6301    DOI : 10.1128/AEM.70.10.6299-6301.2004
Rosebrough SF , Hartley DF 1996 Biochemical modification of streptavidin and avidin: in vitro and in vivo analysis. J. Nucl. Med. 37 1380 - 1384
Salmain M , Ghasemi M , Boujday S , Spadavecchia J , Techer C , Val F 2011 Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level. Biosens. Bioelectron. 29 140 - 144    DOI : 10.1016/j.bios.2011.08.007
Sankaran S , Panigrahi S , Mallik S 2011 Olfactory receptor based piezoelectric biosensors for detection of alcohols related to food safety applications. Sensors Actuat B Chem. 155 8 - 18    DOI : 10.1016/j.snb.2010.08.003
Schlosser G , Kacer P , Kuzma M , Szilagyi Z , Sorrentino A , Manzo C 2007 Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B. Appl. Environ. Microbiol. 73 6945 - 6952    DOI : 10.1128/AEM.01136-07
Seki M , Yamashita Y , Torigoe H , Tsuda H , Sato S , Maeno M 2005 Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae. J. Clin. Microbiol. 43 1581 - 1586    DOI : 10.1128/JCM.43.4.1581-1586.2005
Seo KH , Brackett RE , Hartman NF , Campbell DP 1999 Development of a rapid response biosensor for detection of Salmonella typhimurium. J. Food Prot. 62 431 - 437
Sharma H , Mutharasan R 2013 Review of biosensors for foodborne pathogens and toxins. Sensors Actuat. B Chem. 183 535 - 549    DOI : 10.1016/j.snb.2013.03.137
Shi XM , Long F , Suo B 2010 Molecular methods for the detection and characterization of foodborne pathogens. Pure Appl. Chem. 82 69 - 79    DOI : 10.1351/PAC-CON-09-02-07
Si CY , Ye ZZ , Wang YX , Gai L , Wang JP , Ying YB 2011 Rapid detection of Escherichia coli O157:H7 using surface plasmon resonance (SPR) biosensor. Spectrosc. Spectral Anal. 31 2598 - 2601
Simpkins SA , Chan AB , Hays J , Popping B , Cook N 2000 An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica. Lett. Appl. Microbiol. 30 75 - 79    DOI : 10.1046/j.1472-765x.2000.00670.x
Singh C , Agarwal GS , Rai GP , Singh L , Rao VK 2005 Specific detection of Salmonella Typhi using renewable amperometric immunosensor. Electroanalysis 17 2062 - 2067    DOI : 10.1002/elan.200403334
Song TY , Toma C , Nakasone N , Iwanaga M 2005 Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method. FEMS Microbiol. Lett. 243 259 - 263    DOI : 10.1016/j.femsle.2004.12.014
Spangler BD , Wilkinson EA , Murphy JT , Tyler BJ 2001 Comparison of the Spreeta (R) surface plasmon resonance sensor and a quartz crystal microbalance for detection of Escherichia coli heat-labile enterotoxin. Anal. Chim. Acta 444 149 - 161    DOI : 10.1016/S0003-2670(01)01156-4
Stevens KA , Jaykus LA 2004 Bacterial separation and concentration from complex sample matrices: a review. Crit. Rev. Microbiol. 30 7 - 24    DOI : 10.1080/10408410490266410
Tokarskyy O , Marshall DL 2008 Immunosensors for rapid detection of Escherichia coli O157 : H7-perspectives for use in the meat processing industry. Food Microbiol. 25 1 - 12    DOI : 10.1016/
Tsaloglou MN , Bahi MM , Waugh EM , Morgan H , Mowlem M 2011 On-chip real-time nucleic acid sequence-based amplification for RNA detection and amplification. Anal. Methods 3 2127 - 2133    DOI : 10.1039/c1ay05164d
Velusamy V , Arshak K , Korostynska O , Oliwa K , Adley C 2010 An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnol. Adv. 28 232 - 254    DOI : 10.1016/j.biotechadv.2009.12.004
Vernozy-Rozand C , Mazuy-Cruchaudet C , Bavai C , Richard Y 2004 Comparison of three immunological methods for detecting staphylococcal enterotoxins from food. Lett. Appl. Microbiol. 39 490 - 494    DOI : 10.1111/j.1472-765X.2004.01602.x
Verstraete K , Robyn J , Del-Favero J , De Rijk P , Joris MA , Herman L 2012 Evaluation of a multiplex-PCR detection in combination with an isolation method for STEC O26, O103, O111, O145 and sorbitol fermenting O157 in food. Food Microbiol. 29 49 - 55    DOI : 10.1016/
Vo-Dinh T , Griffin G , Stokes DL , Wintenberg A 2003 Multi-functional biochip for medical diagnostics and pathogen detection. Sensors Actuat B Chem. 90 104 - 111    DOI : 10.1016/S0925-4005(03)00048-0
Wang CH , Lien KY , Wu JJ , Lee GB 2011 A magnetic bead-based assay for the rapid detection of methicillinresistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification. Lab. Chip 11 1521 - 1531    DOI : 10.1039/c0lc00430h
Wang DG , Wang YZ , Wang JH , Zhang XW , Xiao FG 2011 Rapid detection of viable Listeria monocytogenes in raw milk using loop-mediated isothermal amplification with the aid of ethidium monoazide. Milchwissenschaft Milk Sci. Int. 66 426 - 429
Wang LX , Li Y , Mustapha A 2007 Rapid and simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in ground beef by multiplex realtime PCR and immunomagnetic separation. J. Food Prot. 70 1366 - 1372
Wang YX , Ye ZZ , Si CY , Ying YB 2011 Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance. Sensors 11 2728 - 2739    DOI : 10.3390/s110302728
Waswa J , Irudayaraj J , DebRoy C 2007 Direct detection of E. coli O157:H7 in selected food systems by a surface plasmon resonance biosensor. LWT Food Sci. Technol. 40 187 - 192    DOI : 10.1016/j.lwt.2005.11.001
Waswa JW , Debroy C , Irudayaraj J 2006 Rapid detection of Salmonella enteritidis and Escherichia coli using surface plasmon resonance biosensor. J. Food Process Eng. 29 373 - 385    DOI : 10.1111/j.1745-4530.2006.00071.x
World Health Organization. 2007 Food safety and foodborne illness. [Online.]
Wu VCH , Chen SH , Lin CS 2007 Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance. Biosens. Bioelectron. 22 2967 - 2975    DOI : 10.1016/j.bios.2006.12.016
Yamazaki W , Ishibashi M , Kawahara R , Inoue K 2008 Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus. BMC Microbiol. 8
Yamazaki W , Kumeda Y , Uemura R , Misawa N 2011 Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples. Food Microbiol. 28 1238 - 1241    DOI : 10.1016/
Yang H , Ma XY , Zhang XZ , Wang Y , Zhang W 2011 Development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus aureus in food. Eur. Food Res. Technol. 232 769 - 776    DOI : 10.1007/s00217-011-1442-8
Yang L , Bashir R 2008 Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria. Biotechnol. Adv. 26 135 - 150    DOI : 10.1016/j.biotechadv.2007.10.003
Ye YX , Wang B , Huang F , Song YS , Yan H , Alam MJ 2011 Application of in situ loop-mediated isothermal amplification method for detection of Salmonella in foods. Food Control 22 438 - 444    DOI : 10.1016/j.foodcont.2010.09.023
Yoo JH , Choi SM , Choi JH , Kwon EY , Park C , Shin WS 2008 Construction of internal control for the quantitative assay of Aspergillus fumigatus using real-time nucleic acid sequence-based amplification. Diagn. Microbiol. Infect. Dis. 60 121 - 124    DOI : 10.1016/j.diagmicrobio.2007.08.001
Zhao XH , Li YM , Wang L , You LJ , Xu ZB , Li L 2010 Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples. Mol. Biol. Rep. 37 2183 - 2188    DOI : 10.1007/s11033-009-9700-6
Zhao XH , Wang L , Chu J , Li YY , Li YM , Xu ZB 2010 Development and application of a rapid and simple loopmediated isothermal amplification method for food-borne Salmonella detection. Food Sci. Biotechnol. 19 1655 - 1659    DOI : 10.1007/s10068-010-0234-4
Zhao XH , Wang L , Chu J , Li YY , Li YM , Xu ZB 2010 Rapid detection of Vibrio parahaemolyticus strains and virulent factors by loop-mediated isothermal amplification assays. Food Sci. Biotechnol. 19 1191 - 1197    DOI : 10.1007/s10068-010-0170-3
Zhao XH , Wang L , Li YM , Xu ZB , Li L , He XW 2011 Development and application of a loop-mediated isothermal amplification method on rapid detection of Pseudomonas aeruginosa strains. World J. Microbiol. Biotechnol. 27 181 - 184    DOI : 10.1007/s11274-010-0429-0