P1: SFK/UKS BLBS102-c44 P2: SFK BLBS102-Simpson 846 March 21, 2012 14:34 Trim: 276mm X 219mm Printer Name: Yet to Come Part 8: Food Safety and Food Allergens good information into the detail of the technology Whereas, its underlying principle is based on standard PCR, a fluorescent tag is added to the primers so that the amplicons can be detected on a real-time basis by monitoring fluorescence Thus, a detector in the thermocycler detects amplified product as it is produced Real-time PCR protocols can be designed to detect multiple products simultaneously using a similar approach to multiplex PCR—typically, four or five fluorescent tags can be detected simultaneously as with conventional multiplex PCR It also means that products of the similar size can be amplified easily without the challenges posed by separation on a standard electrophoresis gel, as each amplicon will be amplified with a different fluorescent marker The advantages of real-time PCR over conventional PCR include: easy resolution of product, a faster run time as there is no post-PCR gel analysis, and increased sensitivity The latter is greater than conventional PCR, as real-time PCR technology relies on the detection of a signal and its quantification with the release of light being in proportion to amplified product formed Fluorescent dyes such as HEX, FAM, ROX, and SYBR green are some of the commonly available fluorescent dyes, which can be used simultaneously in a multiplex real-time-PCR protocol (Huang et al 2007, Wang et al 2007, Nde et al 2008b) Multiple commercial thermocyclers are available for use in performing real-time PCR offering capabilities from single tubes to 96 and 384 well formats and have the capability to detect up to five different fluorescent targets simultaneously Despite the practical advantages of this method, the expense of the requisite thermocyclers is high that may curtail the use of real-time PCR in some instances Potential applications of real-time PCR include detection of genes or strains in a range of media (Rodriguez-Lazaro et al 2004, Fakhr et al 2006, Bohaychuk et al 2007, Wang et al 2007, O’Grady et al 2008), and diarrheagenic E coli (Vidal et al 2005) Horsmon et al (2006) used real-time fluorogenic PCR to detect entA, encoding the staphylococcal enterotoxin A (SEA) The method detected SEA at levels as low as 1–13 gene copies Fykse et al (2007) used molecular beacon real-time nucleic acid sequence-based amplification for Vibrio cholerae by detecting the cholera toxin gene (ctxA) and the genes tcpA, toxR, hlyA, and groEL and was able to detect the organism at a level of 50 CFU/mL, the method could also differentiate toxigenic from nontoxigenic Vibrio strains by amplification of the toxin genes tcpA and ctxA Grant et al (2006) modified the real-time PCR technique to simultaneously detect heat stable and labile toxin genes of enterotoxigenic E coli with threshold cycles of 25.2–41.1 A primary limitation of PCR has been that it could not distinguish between live and dead cells However, newly designed PCR protocols can target viable cells by detection of mRNA, which is a marker of viability (Klein and Juneja 1997, McIngvale et al 2002, Morin et al 2004) Overall, PCR has tremendous power as a molecular detection and profiling tool especially as a means to detect a pathogen as well as determine a pathogen’s traits or to define its pathotype It can also be useful for clustering gene traits and can provide significant information when bundled with analysis software to sort organisms into cluster groups or by trait possession (RodriguezSiek et al 2005a, 2005b) NEXT-GENERATION TECHNOLOGIES The application of sensitive and rapid detection technologies has become of paramount interest in advancing methods for rapid detection and identification of pathogens of concern In this section, we review some of the newer technologies emerging for the future and their potential for enhancing methods to detect foodborne pathogens As we write this section, however, the reader should be aware that the technology is rapidly changing, some recent reviews in relation to the use of biosensors include articles by Kaittanis et al (2010), Tallury et al (2010), and Velusamy et al (2010) Two of the overriding factors in sensor detection methodologies are the ability to obtain a result in a faster time frame and a preference to automate the system (Anderson and Taitt 2005) Biosensors are detection devices that use biological molecules to recognize and quantify analytes of interest—these analytes include antibodies, receptors, enzymes, nucleic acids, oligosaccharides, peptides, and so on A recognition event between a biomolecule and an agent (e.g., nucleic acid, receptor, and so on) results in an output that can be measured either optically, chemically, or electronically Biosensors should be able to monitor a sample continuously for analyte and provide a quantitative readout (such as measure of concentration) Adenosine Triphosphate Detection Although not a strict biosensor per se, detection of adenosine triphosphate (ATP) from living cells is used as a means to assess the quality of cleaning or disinfection of surfaces and utensils ATP detection usually involves analysis of a sample swab of an area; the swab is then combined with a mixture of the enzyme luciferase and its substrate luciferin The reaction of luciferin and luciferase is dependent on the presence of ATP to catalyze the reaction, which is only found in living cells The ATP combines with luciferin to form luciferyl adenylate and pyrophosphate Luciferin + ATP → Luciferyl + PPi The luciferyl adenylate reacts with oxygen to form oxyluciferin and adenosine monophosphate with the release of light Luciferyl adenylate + O2 → Oxyluciferin + AMP + Light The enzymatic process results in the release of light, which can be measured and is proportional to the amount of ATP present There are a number of handheld devices that can be used for this type of testing and are available commercially Typical levels of detection are 1–200 CFU ATP assays are nonspecific and in general provide information of a quality nature; it cannot detect for specific pathogens or identify the source of ATP and typically are used as an indicator of hygiene quality or successful sanitization In addition, the system may have errors associated with the sampling technique resulting in falsepositives and false-negatives (Carrick et al 2001) Modifications of ATP detection have incorporated the use of fluorescence for P1: SFK/UKS BLBS102-c44 P2: SFK BLBS102-Simpson March 21, 2012 14:34 Trim: 276mm X 219mm Printer Name: Yet to Come 44 Emerging Bacterial Food-Borne Pathogens and Methods of Detection detection of fluoroimmuno-stained cells; bioluminescence with IMS (Takahashi et al 2000, Tu et al 2000, Squirrel et al 2002) DNA Microarrays DNA microarrays consist of gene probes arrayed on a substrate Such arrays may be limited to select genes of interest or be multigenome-wide in scope Among other things, they can be used for comparative genomic hybridization (also called genomotyping), to study genome-wide gene expression or for the detection of pathogens in a sample They have quickly become a powerful tool in genomic analysis of pathogens The microarray itself (often called a gene chip or biochip) consists of a series of DNA molecules of known sequence called probes that are fixed to a substrate (usually, a special type slide) These probes consist of partial gene sequences, generated from PCR, full-length cDNA, or oligonucleotides (Pagotto et al 2005) of the pathogens of interest Such microarrays can be used to detect a large number of pathogens in a sample or alternatively, they can be used to perform expression studies of the whole genome or select genes; for the purposes of this chapter we will focus on the use of microarrays as a detection technology only; for more detail regarding other applications of microarrays the reader is directed to reviews on the subject A recent review by Ojha and Kostrzynska (2008) highlights the application of microarray technology in the field of veterinary research for pathogen infection investigations, diagnostics, and studies of host pathogen interactions Similar research by Jin et al (2005) used microarray technology to investigate E coli O157:H7 Microarrays can also be used to assess similarities between strains, characterize strains or subtype strains Boyd et al 2003, Gaynor et al (2004), Hain et al (2006), Malik-Kale et al (2007), Parker et al (2007), and Raengpradub et al (2008) have described the use of microarray technology to analyze strains at the genetic level for comparative purposes Volokhov et al (2002), (2003), Chen et al (2005), Reen et al (2005), Garaizar et al (2006), Anjum et al (2007), Yoshida et al (2007), Zhang et al (2007), Batchelor et al (2008) have used microarrays to identify various bacteria to species or subspecies level, detection of virulence or antimicrobial resistance genes and Call et al (2001), Chandler et al (2001), Keramas et al (2004), Kostrzynska and Bachand (2006), Kostic et al (2007), Quinones et al (2007) have used them for detection, identification, and characterization of pathogens in a range of samples Given the unlimited amount of information available, microarrays can be designed and built for a range of purposes such as, determining expression of specific virulence or antimicrobial resistance genes or for more specific processes, such as study of invasion, flagellar production, growth processes, or biofilm production DNA microarrays offer much promise for future studies in understanding pathogens, hosts, and production systems—they can be used to model host–pathogen interactions and the effect of various drugs or vaccines on a host or pathogen Therefore, it is likely that microarray technology will provide needed insight into faster methods for detection and understanding the mechanisms of pathogenesis used by food-borne pathogens that can be exploited to make food safer 847 Immunosensors or Biosensors Biosensors are analytical devices that convert a biological response into an electrical signal Biosensors work on the principle of a biological component that is coupled to a physiochemical type transducer that collects signal and converts it to an electronic readout A range of bioreceptors currently recognized for use in pathogen detection include antibodies, enzymes, nucleic acids, cellular, biomimetic, and bacteriophages (Velusamy et al 2010) Biosensor technology has rapidly expanded in the last 10 years and as this chapter is completed, there will no doubt be many more new applications emerging Some of the more promising technologies at this point are based on fiber optics and electrochemical reactions Velusamy et al (2010) provide a comprehensive overview of sensors for application in pathogen detection Fiber Optic Biosensor Optical fibers for the detection of pathogens work on the principle of a fiber optic taper that sends excitation laser light to a detection surface and receives emitted light As such, fiber optic technology has been reported to be very useful in the detection of food-borne pathogens Optical biosensors measure changes in refractive index, fluorescence emission or quenching, chemiluminescence, and fluorescence energy transfer One such fiber optic biosensor operates by using an antibody sandwich format on optic fiber to detect the capture of antigens using fluorescently tagged conjugates Geng et al (2004) used a fiber optic-based detection system to detect Listeria in mixed cultures, and meat sample enrichments with a detection level as low as 10–103 cells/mL In a similar type approach, Kramer and Lim (2004) developed a rapid automated fiber-based biosensor for the detection of Salmonella in sprout rinse water The sensor was capable of detecting Salmonella when counts as low as 50 CFU/g were inoculated into the seeds Fiber optics have been used in Listeria, Salmonella, E coli O157:H7, and Clostridium botulinum toxin detection (Ogert et al 1992, Strachan and Gray 1995, Simpson and Lim 2005, Ko and Grant 2006) and for the detection of S aureus (Chang et al 1996) Raman and Fourier Transform Spectroscopy This technology is frequently used in whole organism fingerprinting but also has application in analysis of a sample of interest The technique does, however, depend on increasing the population so that there is sufficient amount for biomass analysis Raman spectroscopy is an optical technique that uses light scattering to detect pathogens (Schmilovitch et al 2005) Modifications of Raman spectroscopy using surface enhanced technologies (Grow et al 2003, Kalasinsky et al 2007) have also been developed (see below) Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FTIR) is a nondestructive technique for pathogen detection that measures infrared loss P1: SFK/UKS BLBS102-c44 P2: SFK BLBS102-Simpson March 21, 2012 14:34 848 Trim: 276mm X 219mm Printer Name: Yet to Come Part 8: Food Safety and Food Allergens after passing through a sample, the data collected is analyzed using Fourier transformation and the resulting output results in a spectrum identical to the conventional infrared spectroscopy FTIR has been used to differentiate and quantify Salmonella, E coli O157:H7, and Listeria in various media or substrates (Lin et al 2004, Yu et al 2004, Al-Holy et al 2006) Ellis et al (2002) reported the use of FTIR directly on food to detect unique biochemical fingerprints of pathogens and provided a measure of bacterial loads Surface Plasmon Resonance Surface plasmon resonance (SPR) uses reflectance spectroscopy to detect pathogens The principle of the technique is based on the technology being able to detect minor changes in refractive index, which occur as cells of the target bind to receptors immobilized on a transducer surface Changes in the angle of reflected light are measured as a function of changes in density of the medium versus time SPR sensors have been used by a number of researchers for the detection of a range of pathogens including L monocytogenes, Salmonella, E coli O157:H7, and C jejuni (Koubova et al 2001, Bokken et al 2003, Bhunia et al 2004, Leonard et al 2004, Meeusen et al 2005, Subramanian et al 2006, Taylor et al 2006, Waswa et al 2007) SPR technologies use antibodies that are immobilized on a gold electrode surface that can measure miniscule changes in resonance frequency as a result of antibody antigen binding occurring (Feng 2007) Su and Li (2004) incorporated SPR with a piezoelectric system for the detection of E coli O157:H7 at levels ranging from 103 to 108 cells; a similar approach by Oh et al (2004) could detect S typhimurium at levels ranging from 102 to 109 CFU/mL and Leonard et al (2004, 2005) report detection of 105 cells/mL of L monocytogenes in less than 30 minutes Oh et al (2004) report the development of an SPR chip capable of detecting multiple organisms simultaneously including E coli O157:H7, S typhimurium, Legionella pneumophila, and Yersinia enterocolitica Mass Sensitive Biosensors Mass sensitive biosensors work on the principle of detection of minute changes in mass The analysis depends on the use of piezoelectric crystals that vibrate at a specific frequency, as a result of electrical input at the same frequency The frequency depends on electrical input and the mass of the crystal If the mass of the crystal increases due to binding of agents, the weight of the crystal will change and therefore the oscillation frequency The difference in oscillation results in a change, which can be measured electronically Quartz is one of the most common piezoelectric material and the main types of sensors are quartz crystal microbalance (QCM) or surface acoustic wave (SAW) When the surface of the sensor is coated with an antibody and exposed to liquid containing the target pathogen—the pathogens will bind to the antibody resulting in a change in the weight of the crystal, and a shift in its frequency oscillation QCM have been used in the detection of Salmonella (Su and Li 2005), L monocytogenes (Vaughn et al 2001), and E coli O157:H7 (Berkenpas et al 2006) Modifications of the microbalance principle include piezoelectric excited millimeter sized cantilever and magnetoelastic sensors (Ruan et al 2004, Mutharasan and Campbell 2008) Electronic Nose Sensors Electronic nose technology for pathogen detection works on the principle of detection of volatiles produced by active organisms in a food sample The system is based on adsorption of volatiles to a series of conducting organic polymers; once the volatiles are adsorbed there is a change in the resistance of the sensors that can be equated to the presence of an organism or a population of organisms Balasubramanian et al (2005) used a commercial electronic nose system to detect Salmonella in inoculated beef samples The system was able to differentiate Salmonella contaminated from noncontaminated meat Similar systems have also been used in the detection of E coli O157:H7 Younts et al (2002, 2003) assessed the use of gas sensors for detection of E coli O157 and non-O157 strains Sensitivity and specificity ranged from 41.7 to 50% for nonnormalized data, which contrasted to data normalized using artificial neural network (ANN) classification that increased sensitivity from 91.7% to 100% but specificity ranged from 37.5% to 50% Magan et al (2001) used a commercial system for detection of Pseudomonas and Bacillus species in milk; Muhamed-Tahir and Alocilja (2003) developed a portable sensor system for detection of E coli O157:H7 with a minimum detection limit of 7.8 × 101 CFU/mL Arshak et al (2009) used a series of conducting polymers to detect a range of food-borne pathogens including Salmonella, Bacillus cereus and Vibrio parahaemolyticus, and was able to differentiate each based on its signal Nanotechnology for Pathogen Detection As one of the latest technologies, nanotechnology presents a great opportunity for rapid detection, diagnosis, and identification of pathogens Nanoparticles, in particular, gold and silver have electronic and optical properties that make them useful in next generation detection When the particles are coupled to affinity ligands they can be useful in pathogen detection; for example, gold nanoparticles coupled with specific oligonucleotides can detect complementary DNA Other types of nanoparticles including quantum dots and carbon nanotubes have been used in assays for detection of pathogens, toxins, DNA, and in immunoassay development (Kaittanis et al 2010) Recent articles by Kaittanis et al (2010) and Tallury et al (2010) provide some interesting overviews of nanotechnology applications in pathogen detection SUMMARY Pathogens associated with human disease are constantly changing, what was old is now new and what was new has emerged in new ways not previously recognized as a means to cause disease As fast as the pathogens are emerging, technologies and applications for their detection have become the next race to find the P1: SFK/UKS BLBS102-c44 P2: SFK BLBS102-Simpson March 21, 2012 14:34 Trim: 276mm X 219mm Printer Name: Yet to Come 44 Emerging Bacterial Food-Borne Pathogens and Methods of Detection ultimate technology that can detect all in a user-friendly manner, in as short a time as possible and with the ultimate sensitivity and specificity REFERENCES Aarestrup FM, Engberg J 2001 Antimicrobial resistance of thermophilic Campylobacter Vet Res 32: 311–321 Abeyta C et al 1990 Incidence of motile aeromonads from United States west coast shellfish growing estuaries J Food Prot 53: 849–855 Acheson DW 1999 Foodborne Infections Curr Opin Gastroenterol 15: 538–545 Alarcon B et al 2004 Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescent J Agric Food Chem 52: 7180–7186 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Food Microbiology, Fundamentals, and Frontiers ASM Press, Washington, DC, pp 911–934 Franz E, van Bruggen AH 2008 Ecology of E coli O157:H7 and Salmonella enterica in the primary vegetable production chain Crit Rev Microbiol 34: 143–161 ... 14:34 84 8 Trim: 276mm X 219mm Printer Name: Yet to Come Part 8: Food Safety and Food Allergens after passing through a sample, the data collected is analyzed using Fourier transformation and the... growing estuaries J Food Prot 53: 84 9? ?85 5 Acheson DW 1999 Foodborne Infections Curr Opin Gastroenterol 15: 5 38? ??545 Alarcon B et al 2004 Simultaneous and sensitive detection of three foodborne pathogens... Salmonella in a wide variety of food and food- animal matrices J Food Prot 70: 1 080 –1 087 Bokken G et al 2003 Immunochemical detection of Salmonella group B, D and E using an optical surface Plasmon