foodborne illnesses research essay

EDITORIAL article

Editorial: foodborne pathogens: hygiene and safety.

• Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy

Editorial on the Research Topic Foodborne Pathogens: Hygiene and Safety

Introduction

The foodborne outbreaks occurred in last decades highlight the importance of the development and implementation of preventive measures and programs aiming at ensuring food safety on one hand and constituting a common basis for the hygienic production of food on the other hand. In particular, a farm to fork approach has been applied in all sectors of food production chain in order to improve hygiene and reduce all potential biological hazards. The food supply chain is very complex because of the differences in food composition and processing and this can result in emergence and re-emergence of foodborne pathogens. However, many factors related to an increase in foodborne illness have been reported, such as the change in eating habits and consumer preferences, increased international travels, change in food processing, production and distribution, pathogen adaptation to new environments, acquisition of virulence factors and antimicrobial drug resistance by microorganisms, advances in pathogen detection methods, inadequate sanitation and vector control measures, inadequate public health services, including consumer information ( Smith and Fratamico, 2018 ). This Research Topic titled “Foodborne Pathogens: Hygiene and Safety” focuses on important food safety concerns such as the potential presence of pathogens in food as well as their toxins/metabolites, the resistance to antibiotics or sanitizers, and other virulence characteristics. It includes four reviews and 44 original research papers. The main foodborne pathogens studied herein are: Campylobacter jejuni, Cronobacter sakazakii, Escherichia coli, Listeria monocytogenes, Salmonella spp., and Staphyloccus aureus , but some other researches deal with Helicobacter pilori, Klebsiella pneumoniae, Vibrio parahaemolyticus , mycobacteria, and molds as well. Studies on characterization and genetic typing of foodborne pathogens, detection methods and inactivation of these microorganisms by natural preservatives derived from plant sources, essential oils and biocontrol, and influence of probiotics are also reported.

Prevalence and Monitoring of Pathogens in Food

Foodborne diseases represent one of the most important public health troubles worldwide. The potential of foodborne pathogens to cause illness or even death in consumers highlights the importance of such events and consequent need of their monitoring and prevention. Millions of cases of foodborne illnesses and/or chronic complications are reported in many countries every year ( Heredia and García, 2018 ). Li S. et al. studied the prevalence and characteristics of Non-typhoidal Salmonella isolated from poultry meat (broilers and spent hens) from supermarkets in China. Three serotypes were identified in 40 Salmonella strains and Salmonella Enteritidis resulted as dominant. The antibiotic resistance was tested as well, showing the highest rates to ampicillin for the strains isolated from commercial broilers, and to nalidixic acid for those isolated from spent hence. Thung et al. investigated the prevalence of Salmonella spp. in different beef meat samples from retail markets in Malaysia as well as the virulence genes and antimicrobial resistance. Eight different serovars were identified and Salmonella Agona was the predominant one. All 23 isolates were resistant at least to three antibiotics. Colello et al. determined the prevalence of Salmonella spp. in 764 samples collected from swine farms, slaughterhouses, boning rooms, and retail markets. The strains were classified into five serotypes (i.e., Salmonella Typhimurium, Salmonella Kentucky, Salmonella Brandenburg, Salmonella Livingstone, and Salmonella Agona) and showed different resistance to antibiotics.

The microbiological quality (mesophilic aerobic bacteria, total coliforms, yeasts, and molds) and safety level ( E. coli O157:H7, Shiga toxin-producing E. coli, Salmonella Enteritidis, Salmonella Typhimurium, Listeria spp., and L. monocytogenes ) of organic and conventional vegetables from Malaysia were evaluated. Salmonella spp., L. monocytogenes , and Listeria spp. were the most representatives, with no trend between organically or conventionally grown vegetables ( Kuan et al. ). The presence of total and pathogenic V. parahaemolyticus strains was detected in short mackerel samples collected from different retail markets in Malaysia. The antimicrobial susceptibility profiles were also studied, showing a resistance to penicillin G and ampicillin ( Tan et al. ).

The genetic diversity as well as the antibiotic resistance and biofilm formation of Cronobacter spp. recovered from spices and cereals were studied by Li Y. et al. Cronobacter sakazakii was the most common species, and 62.5% of 40 Cr. sakazakii strains were non-biofilm producers. Parra-Flores et al. evaluated the presence of Cr. sakazakii , microbiological levels of aerobic plate count and Enterobacteriaceae in dairy product batches associated with a recent food alert in Chile.

Sevilla et al. investigated the presence of members of the genus Mycobacterium by culture and PCR-based methods in raw dairy and meat products purchased at different supermarkets in Spain. Mycobacterial DNA was detected in 23 out of 257 samples, corresponding to Mycobacterium avium, Mycobacterium tuberculosis , and other non-tuberculous mycobacteria.

Wang W et al. submitted two papers to this Research Topic, the first one concerned the complete genomic analysis of a Salmonella Typhimurium isolate from ready-to-eat pork samples in China, the second dealt with the prevalence of S. aureus among raw milk from dairy cows with clinical mastitis.

Lipophilic marine biotoxins belonging to okadaic acid, pectenotoxin, yessotoxin, and azaspiracid groups were determined in specimens of mussels collected along the coasts of the Central Adriatic Sea (Italy) by LC-MS/MS. The concentrations exceeded the maximum regulatory limits only for 11 out of 400 samples, and some samples showed a multi-toxin mixture contamination ( Schirone et al. ).

Antimicrobial Resistance and Virulence Factors

Microbial interactions can show beneficial or detrimental effects that influence the fate of pathogenic species contaminating foods. The study of such interactions can provide a new knowledge about the different activities of the microorganisms from proliferation and metabolism to pathogenicity and virulence ( Zilelidou and Skandamis, 2018 ).

Dairy products can host microorganisms belonging to Enterobacteriaceae family showing multidrug resistance to antibiotics and other virulence factors such as production of biofilm and synthesis of proteolytic and lipolytic enzymes responsible for spoilage. Their presence can be reduced or avoided through good hygiene conditions during processing and manufacturing, as well as storage and distribution ( Amorim et al. ). Chagnot et al. investigated the adhesion of E. coli O157:H7 to well-defined types of skeletal muscle and demonstrated that such microorganism mainly adhered to the extracellular matrix of muscle cells, with no significant differences among the different constituent myofibres, whereas the influence of post-mortem structural modifications of muscle tissues was substantial.

The adhesion capacity of 40 C. jejuni strains to abiotic surfaces was studied. All C . jejuni strains were shown to be capable of forming strong biofilms when Mueller Hinton medium was supplemented with chicken juice. However, the use of biocides was effective in controlling viable cells of strains in biofilm ( Melo et al. ). Oh et al. demonstrated that ferrous and ferric iron stimulated biofilm formation in C. jejuni through oxidative stress. Premarathne et al. determined the prevalence and antibiotic resistance of Campylobacter spp. in the beef food system in Malaysia. Most isolates were identified as C. jejuni , with a high percentage resistant to tetracycline and ampicillin.

The effect of cold stress on the adhesion to abiotic surfaces and biofilm formation of 22 L. monocytogenes strains from different serogroups and origins was studied by Lee et al. Such study demonstrated the increase of the adhesion capacity, whereas the cold-adapted cells remained in planktonic form. Pasquali et al. studied the persistence and physiological adaptation to food-processing environmental stress of L. monocytogenes strains from a rabbit meat processing plant. While some strains showed a resistance to sanitizers, some others were biofilm producers and these specific characteristics could contribute to their high prevalence. The nucleotide diversity of L. monocytogenes strains from human clinical cases—as well as food or food-related environments originating from three different geographical locations (i.e., Australia, Greece, and Ireland)—was studied by Poimenidou et al. The authors demonstrated that virulence genes showed different evolutionary pathways affected by the origin and serotype of the specific strain.

Lang et al. demonstrated that drying of milk powder increased the Caco-2 cell invasion capacity of two pathogens, i.e., Salmonella enterica and Cr. sakazakii , probably due to the activation of stress response transcriptional factors, and a subsequent heat treatment did not offset the loss of cultivability that was observed in the experimental design.

Javed et al. described the characteristics, prevalence, survival, and transmission, as well as pathogenesis and virulence determinants of Helicobacter pullorum . Such microorganism causes gastroenteritis in poultry, but it is also an emerging zoonotic bacterium associated with enteric infections in humans with colitis, hepatitis, and recurrent diarrhea.

Detection Methods Applicable in Food Industry

A microfluid system combining loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples was performed. Such method showed relative sensitivity, specificity and accuracy of 100% and could be used in the food industry as a simple, inexpensive and fast analytical approach ( Garrido-Maestu et al. ). A new standard operating procedure for multiple-locus variable number tandem repeat analysis (MLVA) of Salmonella Dublin was proposed by Vignaud et al. The MLVA scheme was applied to a foodborne outbreak occurred in France in 2012, in order to discriminate between epidemiologically related strains and sporadic case strains. Fong et al. characterized four Salmonella phages isolated from irrigation water, cattle feces, and sediment from irrigation ditches, based on their phenotypic and genotypic determinants, and assessed their infectivity against various Salmonella strains in vitro . Among them, the phage isolate SI1 was the most effective in control of Salmonella Enteritidis in sprouting alfalfa seeds artificially contaminated.

The study of Ogrodzki and Forsythe described the application of three genotyping methods (Multilocus Sequence Typing, MLST, capsular profiling of the K -antigen and colanic acid byosinthesis regions and CRISPR- cas array profiling) to discriminate different species belonging to Cronobacter genus. Chase et al. found a Cr. sakazakii isolate, H322, in a batch of powdered infant formula (PIF) and two other isolates showing indistinguishable Pulsed Field Gel Electrophoresis patterns with H322, during routine testing of these products ready for distribution. Therefore, whole genome sequencing, as well as microarray analysis, was applied to these strains, showing a phylogenetic relation among them. This study confirmed that the pathogen could persist within the PIF manufacturing facility for years.

Wang J. et al. developed a novel approach to predict the growth kinetics of S. aureus on rice cake under different environmental conditions. These probability models could be useful for food safety management and microbiological risk assessment of such pathogen.

Listeria monocytogenes encodes a functional ArgR, a transcriptional regulator with specific functions in arginine metabolism regulation and acid tolerance. Cheng et al. showed that a single ArgR regulator could have opposite regulatory effects on the arginine deiminase pathway in an arginine-independent and dependent manner under neutral and acidic conditions, respectively.

Henri et al. compared different genomic methods, i.e., MLST, Whole Genome Sequencing (WGS), and Single Nucleotide Polymorphism (SNP), used to cluster L. monocytogenes strains. This study revealed high concordance between MLST and SNP approaches for diagnostic laboratories responsible for outbreak detection and surveillance.

Williams et al. described a rapid flow cytometric method for determining E. coli O157:H7 contamination in raw spinach. This method could be used as a screening tool to detect such microorganism in food. The presence of two distinct loci of heat resistance on a plasmid encoding type three fimbriae and three bacteriocins, in 1 out 90 E. coli raw milk cheese strains, was investigated. Such plasmid was transferable to other E. coli strains including Shiga-toxin-producing strains, posing great concern in food production environments ( Boll et al. ). Hussain et al. evaluated the contamination with pathogenic and/or multiresistant E. coli among broiler free-range chicken specimens (ceca and meat). The isolates were characterized using both conventional typing and WGS and compared with human E. coli pathotypes. The results showed that the poultry E. coli strains shared closer genetic identity to human E. coli . Zhang B. et al. demonstrated that a specific genetic marker (named fimbrial gene z3276 ) of Enterohemorrhagic E. coli O157:H7 encoded multifunctional structures with properties contributing to host colonization and bacterial survival in the environment.

The regulatory mechanism of secondary metabolism by comparative transcriptomic in Aspergillus flavus was studied by Yao et al. Such approach allowed the authors to identify known and novel regulators required for aflatoxins biosynthesis.

Zhang S. et al. determined biotypes, serotypes, virulence genes, and antimicrobial resistance patterns of K. pneumoniae strains from retail foods in China. The authors reported that some strains from the same geographic area had a closer relationship and they showed high levels of resistance to ampicillin.

Yang et al. utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in Trichinella spiralis .

Zhao et al. described the surface enhanced Raman spectroscopy as testing technology used for the detection of pathogenic bacteria in food. Such method can be considered fast, simple, specific, and sensitive.

Promising Strategies for Food Preservation

Preservation technologies are applied to extend the shelf-life, improve the hygienic quality, and ensure the safety of food. In food industry bacteriocins or other natural preservatives such as herbal extracts and essential oils are used as alternative to prevent the growth of both pathogenic and spoiling microorganisms ( Martínez et al., 2019 ; Nazari et al., 2019 ).

Gray et al. described novel biocontrol methods such as bacteriophages, endolysins, bacteriocins, and plant derived products (essential oils) for the prevention of biofilm formation by L. monocytogenes in food production facilities. The inhibitory effect of Hedychium spicatum L. essential oil and radiation on production of deoxynivalenol and zearalenone by Fusarium graminearum in maize grains was studied by response surface methodology. The results showed a reduction of fungal growth rate as well as mycotoxin content ( Kalagatur et al. ).

Bajpai et al. described a significant antibacterial activity of a quinoline compound (jineol) isolated from the insect Scolopendra subspinipes mutilans against two selected foodborne pathogens (i.e. E. coli O157:H7 and S. aureus KCTC-1621). Such compound could be used as alternative means of antimicrobial in pharma and food industries.

The study of García and Cabo focused on the optimization of E. coli inactivation by a quaternary ammonium compound based on a mathematical model. The results showed that the optimal disinfectant dose increased exponentially with the initial bacterial concentration.

Different pressure-temperature combinations were applied to investigate the inactivation kinetics of E. coli, Listeria innocua , and S. aureus in black tiger shrimp. Staphylococcus aureus was the most baro-resistant species among the three bacteria. Such study could be used to predict non-linear survival curves of other microorganisms in foods ( Kaur and Rao ).

In their study, Kiran et al. isolated an actinobacterial strain from a marine sponge producing a lipopeptide that was demonstrated to be an effective emulsifier as well as good antioxidant and protective agent against S. aureus . The authors used this lipopeptide as food additive in muffin production with good results in organoleptic qualities of such food.

Kollanoor Johny et al. evaluated the antimicrobial effects of subinhibitory concentrations of two plant-derived compounds (i.e., trans -cinnamaldehyde and eugenol) on different genes of S. enterica serotype Enteritidis phage type 8 associated with virulence, colonization, motility, and invasion capability of such pathogen.

Mohanta et al. described the biological synthesis of silver nanoparticles using a cell-free aqueous leaf extract of plant Protium serratum and their antibacterial activity against some foodborne pathogens, i.e., S. aureus, E. coli , and Pseudomonas aeruginosa . The authors suggested the application of such nanoparticles in food packaging materials as well as disinfectant and cleaning agents.

Nair and Kollanoor Johny submitted two papers to the present Research Topic, the first study described the potential of pimenta leaf essential oil in reducing Salmonella Heidelberg attachment on to turkey skin during poultry processing, whereas the second work studied the antimicrobial function of a dairy-originated probiotic strain against multidrug resistant Salmonella Heidelberg in poults, i.e., young turkeys. The cecal colonization, dissemination to internal organs and potential for skeletal muscle deposition of multidrug resistant strains of Salmonella Heidelberg were studied by a challenge experimental design in poults and adult turkey hens. The results showed the highest recovery in the cecum followed by spleen, liver, thigh, and breast, and could be used to better control this microorganism at farm level and improve the safety of turkey products ( Nair et al. ).

Conclusions

The high number of studies collected in this Research Topic confirms the importance of foodborne pathogens as a global issue and provides a robust and up-to-date scientific advice. It has been highlighted how much important and essential is a rapid detection of foodborne pathogens by sensitive culture independent methods and by new technologies such as WGS or other biomarkers assay analysis. The outbreak investigations play also key roles in the prevention of foodborne pathogens growth and diffusion, such as their food vehicles and how the contamination can occur in the food supply chain. The positive results of this Research Topic suggest to collect additional and new data for the future on this topic “Foodborne pathogens: hygiene and safety.”

Author Contributions

MS and PV drafted the editorial. RT and GS contributed to editorial revision. All authors approved the final paper.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Heredia, N., and García, S. (2018). Animals as sources of food-borne pathogens: a review. Anim. Nutr . 4, 250–255. doi: 10.1016/j.aninu.2018.04.006

PubMed Abstract | CrossRef Full Text | Google Scholar

Martínez, B., García, P., and Rodríguez, A. (2019). Swapping the roles of bacteriocins and bacteriophages in food biotechnology. Curr. Opin. Biotechnol . 56, 1–6. doi: 10.1016/j.copbio.2018.07.007

Nazari, M., Ghanbarzadeh, B., Kafil, H. S., Zeinali, M., and Hamishehkar, H. (2019). Garlic essential oil nanophytosomes as a natural food preservatives: its application in yogurt as food model. Colloid Interface Sci. Commun . 30:100176. doi: 10.1016/j.colcom.2019.100176

CrossRef Full Text | Google Scholar

Smith, J. L., and Fratamico, P. M. (2018). Emerging and re-emerging foodborne pathogens. Foodborne Pathog. Dis. 15, 737–757. doi: 10.1089/fpd.2018.2493

Zilelidou, E. A., and Skandamis, P. N. (2018). Growth, detection and virulence of Listeria monocytogenes in the presence of other microorganisms: microbial interactions from species to strain level. Int. J. Food Microbiol . 277, 10–25. doi: 10.1016/j.ijfoodmicro.2018.04.011

Keywords: food, microorganisms, virulence, illness, preservatives

Citation: Schirone M, Visciano P, Tofalo R and Suzzi G (2019) Editorial: Foodborne Pathogens: Hygiene and Safety. Front. Microbiol. 10:1974. doi: 10.3389/fmicb.2019.01974

Received: 29 June 2019; Accepted: 12 August 2019; Published: 27 August 2019.

Reviewed by:

Copyright © 2019 Schirone, Visciano, Tofalo and Suzzi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Maria Schirone, mschirone@unite.it

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

• Skip to main content
• Skip to FDA Search
• Skip to in this section menu
• Skip to footer links

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you're on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

U.S. Food and Drug Administration

•   Search
•   Menu
• Science & Research (Food)
• Social and Behavioral Science Research (Food)

Consumer Research on Foodborne Illness

Below are research abstracts of consumer research studies conducted or supported by the Center for Food Safety and Applied Nutrition.

The FDA Food Safety Survey: A Data Resource

The U.S. Food and Drug Administration (FDA) Food Safety Survey is a single-stage, random-digit-dialing tracking survey of a nationally representative sample of American consumers. Data were collected in 1988, 1993, 1998, 2001, 2006, and 2010 with sample sizes of 3,202, 1,620, 2,001, 4,482, 4,539, and 4,568, respectively. The purpose of the survey is to track American consumers’ knowledge, behavior, and perceptions on a number of food-safety related topics. These topics include 1) perception of individual and societal risk related to food consumption, 2) food handling, 3) food product safety label understanding and usage, 4) consumption of potentially risky foods, 5) attitude toward new food technologies, 6) perception, knowledge, and experience with foodborne illness, 7) food safety knowledge sources, and 8) consumers’ experience with food allergies. In addition, each wave queries consumers on recent FDA food safety advisories and other topics of current interest to the FDA. The available demographic information includes gender, age, education, race/ethnicity, household size, health status, region, and household income. [Contact: Amy Lando ]

Consumers’ Use of Personal Electronic Devices in the Kitchen. 2018. Amy M. Lando, Michael C. Bazaco and Yi Chen. Journal of Food Protection.

Smartphones, tablets, and other personal electronic devices have become ubiquitous in Americans’ daily lives. These devices are used by people throughout the day, including while preparing food. For example, a device may be used to look at recipes and therefore be touched multiple times during food preparation. Previous research has indicated that cell phones can harbor bacteria, including opportunistic human pathogens such as Staphylococcus and Klebsiella spp. This investigation was conducted with data from the 2016 Food Safety Survey (FSS) and from subsequent focus groups to determine the frequency with which consumers use personal electronic devices in the kitchen while preparing food, the types of devices used, and hand washing behaviors after handling these devices. The 2016 FSS is the seventh wave of a repeated cross-sectional survey conducted by the U.S. Food and Drug Administration in collaboration with the U.S. Department of Agriculture. The goal of the FSS is to evaluate U.S. adult consumer attitudes, behaviors, and knowledge about food safety. The FSS included 4,169 adults that were contacted using a dual-frame (land line and cell phone interviews) random-digit-dial sampling process. The personal electronics module was the first of three food safety topics discussed by each of eight consumer focus groups, which were convened in four U.S. cities in fall 2016. Results from the 2016 FSS revealed that of those individuals who use personal electronic devices while cooking, only about one third reported washing hands after touching the device and before continuing cooking. This proportion is significantly lower than that for self-reported hand washing behaviors after touching risky food products such as raw eggs, meat, chicken, or fish. Results from the focus groups highlight the varied usage of these devices during food preparation and the related strategies consumers are using to incorporate personal electric devices into their cooking routines. [Contact: Amy Lando ]

Mapping Sources of Food Safety Information for U.S. Consumers: Findings From a National Survey 2016. Xiaoli Nan, Linda Verrill, and Jarim Kim. Health Communication.

This research examines the sources from which U.S. consumers obtain their food safety information. It seeks to determine differences in the types of information sources used by U.S. consumers of different sociodemographic background, as well as the relationships between the types of information sources used and food safety risk perceptions. Analyzing the 2010 Food Safety Survey (N = 4,568) conducted by the U.S. Food and Drug Administration, we found that age, gender, education, and race predicted the use of different sources for food safety information. Additionally, use of several information sources predicted perceived susceptibility to foodborne illnesses and severity of food contamination. Implications of the findings for food safety risk communication are discussed. [Contact: Linda Verrill ]

Importance of Cohorts in Analyzing Trends in Safe At-Home Foodhandling Practices. 2015. Mario Teisl, Amy M. Lando, Alan S. Levy, and Caroline L. Noblet. Food Control 62: 381-389.  

Safe in-home food preparation is the last line of defense for preventing foodborne illness. The Food Safety Survey assessing consumers' food handling behavior has been conducted every 3-5 years (1993, 1998, 2001, 2006, 2010) using a random digit telephone sample of United States adult consumers. Sample sizes ranged from 1620 to 4547. A previous analysis of this data has examined trends in safe food handling (as measured by washing hands and/or cutting boards after touching/cutting raw meat or chicken and by washing hands after cracking eggs). We continue and expand this analysis by modeling the unique effects of age, survey period (year) and birth cohort on safe food handling. We find that age, period, and cohort effects are relevant in measuring changes in food handling behavior; however, the effects are not similar in size or apparent mediating process. The strongest effect is period, followed by age and cohort. Thus it appears contemporaneous changes in information activity can make relatively large short-run improvements, whereas changes in one's maturation and accumulated experience have quadratic effects, and the unique shared experience of cohort leaves its own definite long-lasting imprint. We propose that the birth cohort effects can be explained by the food safety environment during young adulthood. Those who were young adults in two critical time periods – before 1940 when there were widespread foodborne infections and immediately after the 1993 outbreak of Escherichia coli O157:H7 – have better food handling behaviors. [Contact: Amy Lando ]

Self-Reported Hand Washing Behaviors and Foodborne Illness: A Propensity Score Matching Approach. 2014. Mir Ali, Linda Verrill, and Yuanting Zhang. Journal of Food Protection 77(3):352-358. 

Hand washing is a simple and effective but easily overlooked way to reduce cross-contamination and the transmission of foodborne pathogens. In this study, we used the propensity score matching methodology to account for potential selection bias to explore our hypothesis that always washing hands before food preparation tasks is associated with a reduction in the probability of reported foodborne illness. Propensity score matching can simulate random assignment to a condition so that pretreatment observable differences between a treatment group and a control group are homogenous on all the covariates except the treatment variable. Using the U.S. Food and Drug Administration's 2010 Food Safety Survey, we estimated the effect of self-reported hand washing behavior on the probability of self-reported foodborne illness. Our results indicate that reported washing of hands with soap always before food preparation leads to a reduction in the probability of reported foodborne illness. [Contact: Linda Verrill ]

Consumer Vegetable and Fruit Washing Practices in the United States, 2006 and 2010. 2012. Linda Verrill, Amy M. Lando, and Kellie M. O'Connell. Food Protection Trends 32(4):164-172. 

Vegetables and fruits may become contaminated with pathogens anywhere along the farm-to-plate continuum. There-fore, the FDA recommends that vegetables and fruits that have not already been washed be washed by the consumer before slicing or consuming them. The FDA included in its 2006 and 2010 Food Safety Survey a series of questions about purchasing and washing of strawberries, tomatoes, cantaloupes, and bagged, pre-cut lettuce. The Food Safety Survey is a telephone survey tracking consumers' knowledge, attitudes and behaviors related to food safety. In 2006, of those who buy these products, 98% wash strawberries, 97% wash tomatoes, 57% wash cantaloupes and 54% wash bagged pre-cut lettuce. Overall, for both years, more women than men wash cantaloupes, and more men than women wash bagged pre-cut lettuce. Cantaloupe washing declined from 2006 to 2010 for men, while lettuce washing increased for women in the same period. Targeted education campaigns should emphasize the importance of washing produce, especially fruits with hard rinds. [Contact: Linda Verrill ]

Trends in Ownership and Usage of Food Thermometers in the United States, 1998 through 2010. 2012. Amy M. Lando and Cary C. Chen. Journal of Food Protection 75(3):556-562.

Food safety research has shown that the use of a food thermometer is the best way to ensure that meat, poultry, and other foods reach an internal temperature sufficient to destroy foodborne pathogens. The 1998, 2001, 2006, and 2010 Food Safety Surveys were used to analyze changes in food thermometer ownership and usage for roasts, chicken parts, and hamburgers in the United States. A probit regression model was used to evaluate differing trends in ownership across demographic subgroups, and probit models with sample selection were used to evaluate differing trends in food thermometer usage for roasts, chicken parts, and hamburgers. The Food Safety Surveys are nationally representative telephone surveys tracking consumers' food safety attitudes and behaviors. Findings from these surveys indicate that the percentage of consumers who own food thermometers has increased from 49% in 1998 to 70% in 2010 (P < 0.05). The use of food thermometers has also increased over this time period but varies by food type. Of those who own food thermometers, a higher percentage reported using thermometers for roasts (76% in 1998 and 82% in 2010, P < 0.05) than for chicken parts (33% in 1998 and 53% in 2010, P < 0.05) and hamburgers (14% in 1998 and 23% in 2010, P < 0.05). The results also show that men, non-Hispanic whites, those with some college education or higher, those with higher incomes, and those 65 years and older were more likely to own food thermometers. After controlling for food thermometer ownership, those aged 18 to 29 years were more likely to use a food thermometer for roasts and chicken parts than those aged 65 to 101 years. The results suggest that educational programs encouraging food thermometer usage should focus first on food thermometer ownership. [Contact: Amy Lando ]

Trends in U.S. Consumers’ Safe Handling and Consumption of Food and Their Risk Perceptions, 1988 through 2010. 2011. Sara B. Fein, Amy M. Lando, Alan S. Levy, Mario F. Teisl, and Caroline Noblet. Journal of Food Protection 74(9):1513-1523 . 

Although survey results measuring the safety of consumers’ food handling and risky food consumption practices have been published for over 20 years, evaluation of trends is impossible because the designs of published studies are not comparable. The Food Safety Surveys used comparable methods to interview U.S. adults by telephone in 1988, 1993, 2001, 2006, and 2010 about food handling (i.e., cross-contamination prevention) and risky consumption practices (eating raw or undercooked foods from animals) and perceived risk from foodborne illness. Sample sizes ranged from 1,620 to 4,547. Responses were analyzed descriptively, and four indices measuring meat, chicken, and egg cross-contamination, fish cross-contamination, risky consumption, and risk perceptions were analyzed using generalized linear models. The extent of media coverage of food safety issues was also examined. We found a substantial improvement in food handling and consumption practices and an increase in perceived risk from foodborne illness between 1993 and 1998. All indices were stable or declined between 1998 and 2006. Between 2006 and 2010, the two safe food handling practice indices increased significantly, but risk perceptions did not change, and safe consumption declined. Women had safer food handling and consumption practices than men. The oldest and youngest respondents and those with the highest education had the least safe food handling behaviors. Changes in safety of practices over the survey years are consistent with the change in the number of media stories about food safety in the periods between surveys. This finding suggests that increased media attention to food safety issues may raise awareness of food safety hazards and increase vigilance in food handling by consumers. [Contact: Amy Lando ]

Developing Consumer-focused Risk Communication Strategies Related to Food Terrorism. 2011. Sara Eggers, Linda Verrill, Cory M. Bryant, and Sarah L. Thorne. International Journal of Food Safety, Nutrition and Public Health 4(1):45-62.  

Risk communication strategies related to food terrorism (FT) threats should reflect an in-depth understanding of consumers’ perceptions, priorities, and information needs related to those threats. To support development of communication strategies, we used a mental models approach to risk communication method to design, conduct and analyse 50 semi-structured telephone interviews with US adults. Interviewees generally lacked well-defined mental models specific to FT, and, instead, drew on their perceptions of terrorism in general, accidental contamination, product recalls, and emergency preparedness. Assessments of their personal threat of FT were influenced by their beliefs about the nature of terrorism, their confidence in government and the food system to prevent and respond to terrorism threats, and their personal control over food choices. These qualitative research results support guidance for developing and implementing consumer-focused FT risk communications strategies. [Contact: Linda Verrill ]

Awareness and Knowledge of Methylmercury in Fish in the United States. 2011. Amy M. Lando, and Yuanting Zhang. Environmental Research 111(3):442-450.

In the 1970s several states in the Great Lakes region became concerned about mercury contamination in lakes and rivers and were the first to issue local fish consumption advisories. In 2001, the Food and Drug Administration (FDA) advised pregnant women, nursing mothers, young children, and women who may become pregnant not to consume shark, swordfish, king mackerel, and tilefish and recommended that these women not exceed 12 ounces of other fish per week. In 2004, FDA reissued this advice jointly with the U.S. Environmental Protection Agency (EPA) and modified it slightly to provide information about consumption of canned tuna and more details about consumption of recreationally caught fish. Though several studies have examined consumers’ awareness of the joint FDA and EPA advisory as well as different state advisories, few used representative data. We examined the changes in awareness and knowledge of mercury as a problem in fish using the pooled nationally representative 2001 and 2006 Food Safety Surveys (FSS) with sample sizes of 4482 in 2001 and 2275 in 2006. Our results indicated an increase in consumers’ awareness of mercury as a problem in fish (69% in 2001 to 80% in 2006, p<.001). In our regression models, we found that in both years, parents having children less than 5 years of age were more aware of mercury in fish and knowledgeable about the information contained in the national advisories about mercury in fish (p<.01) than other adults. In both 2001 and 2006, women of childbearing age (aged 18–45) were less aware and knowledgeable about this information than other women. However, women of all age groups had larger gains in awareness and knowledge than their male counterparts during this time. Participants’ race, education, income, region, fish preparation experiences, having a foodborne illness in the past year, and risk perceptions about the safety of food were significant predictors of their awareness and knowledge. [Contact: Amy Lando ]

Food Safety Perceptions and Practices of Older Adults. 2011. Amy L. Anderson, Linda A. Verrill, and Nadine R. Sahyoun. Public Health Reports 126:220-227.

Older adults are considered more vulnerable to foodborne illness due to lowered immune function. We compared the food safety perceptions and practices of older and younger adults and determined associations with demographic characteristics. We focused on 1,317 participants ≥60 years of age from the U.S. Food and Drug Administration’s 2006 Food Safety Survey, a telephone survey of a nationally representative sample of American consumers. We used data on participants ≥60 years of age to compare younger and older adults, and used Pearson’s Chi-square tests to determine whether perceptions and practices differed by age, gender, level of education, living arrangement, and race/ethnicity. We conducted multiple logistic regression analysis to assess relationships of demographic characteristics and food safety perceptions with food safety practices of older adults. We found that adults ≥60 years of age were more likely to follow recommended food safety practices than those <60 years of age. Sixty-six percent of adults ≥60 years of age reported eating potentially hazardous foods in the past year compared with 81% of adults <60 years of age. Among people ≥60 years of age, women, those with less education, and nonwhite individuals generally had better food safety practices and a greater awareness of food safety risk. These findings suggest that certain subsets of the older adult population are less likely to follow recommended food safety practices and, thus, are at greater risk of foodborne illness. Food safety education for older adults should target men and those with more education and higher incomes. [Contact: Linda Verrill ]

Practice-specific risk perceptions and self-reported food safety practices. 2008. Alan S. Levy, Conrad J. Choinière, and Sara B. Fein. Risk Analysis 28(3):749-761.

The relationship between risk perception and risk avoidance is typically analyzed using self-reported measures. However, in domains such as driving or food handling, the validity of responses about usual behavior is threatened because people think about the situations in which they are self-aware, such as when they encounter a hazard. Indeed, researchers have often noted a divergence between what people say about their behavior and how they actually behave. Thus, in order to draw conclusions about risk perceptions and risk avoidance from survey data, it is important to identify particular cognitive elements, such as those measured by questions about risk and safety knowledge, risk perceptions, or information search behavior, which may be effective antecedents of self-reported safety behavior. It is also important to identify and correct for potential sources of bias that may exist in the data. The authors analyze the Food and Drug Administration's 1998 Food Safety Survey to determine whether there are consistent cognitive antecedents for three types of safe food practices: preparation, eating, and cooling of foods. An assessment of measurement biases shows that endogeneity of food choices affects reports of food preparation. In addition, response bias affects reports of cooling practices as evidenced by its relation to knowledge and information search, a pattern of cognitive effects unique to cooling practices. After correcting for these biases, results show that practice-specific risk perceptions are the primary cognitive antecedents of safe food behavior, which has implications for the design of effective education messages about food safety. [Contact: Amy Lando ]

Consumer Decisions on Storage of Packaged Foods. 2007. Amy M. Lando, and Sara B. Fein. Food Protection Trends 27(5): 307-313.

We investigate the causes of consumer uncertainty regarding storage of packaged foods by examining the characteristics of the consumers, the type of food products and packaging, and the where the product was stored at purchase. Consumers’ self-reported refrigeration practices from the 1998 Food Safety Survey are analyzed descriptively and by logistic regression. Eleven percent of the 2,001 respondents reported difficulty during the past three months in deciding whether to refrigerate a packaged food. When consumers do have difficulty, it is likely that the products either are new to them or need to be stored in an unexpected way. The most likely to report uncertainty about whether to refrigerate were people of middle age and people likely to be more attuned to food safety issues – those who have some college or higher education, who look at many sources of food information, and who thought that a household member had a recent foodborne illness. The results suggest that additional education may be needed to inform consumers about proper refrigeration and that storage information on packages is particularly important for foods that are stored at room temperature until opened but that then need refrigeration. [Contact: Amy Lando ]

Awareness and Perceived Risk of Pesticide and Antibiotic Residues in Food: Socioeconomic Variations Among US Consumers. 2006. Steven T. Yen, Kimberly L. Jensen, and Chung-Tung J. Lin. Food Protection Trends 26(9): 654-661.

We investigate American consumers’ awareness and perceived risk of pesticide and antibiotic residues in food, and how socioeconomic characteristics affect the awareness and risk perception. Based on a 2001 national telephone survey, we employ a statistical approach that accommodates correlations between the two issues and the fact that perceived risk was collected in the survey only from those who were aware of a given issue. Most individuals have heard of pesticide residues but only some have heard of antibiotic residues, though the latter were perceived as a more serious food safety problem than the former. Awareness of one residue problem is associated with a lower perceived risk of the other problem. Income, age, college-or-more education, and being the main meal preparers are related to more awareness of the two issues. Awareness of pesticide residues is lower among Hispanics and blacks, while awareness of antibiotic residues is lower among blacks. Among those who had heard of pesticide residues, the perceived risk of the residues was higher with higher income and being a Midwest resident and lower with a larger number of adults in the household, being a female, older, Hispanic or black, and being a main meal preparer in the household. Among those who had heard of antibiotic residues, the perceived risk of the residues was higher with higher income and being a Hispanic and lower being a female or black. Results also suggest that our statistical approach is worth considering in future research of similar topics. [Contact: Chung-Tung Lin ]

Awareness of Foodborne Pathogens Among US Consumers. 2005. Chung-Tung J. Lin, Kimberly L. Jensen, and Steven T. Yen. Food Quality and Preference 16(5): 401-412.

Each year in the United States, microbial pathogens cause millions of cases of foodborne disease and result in many hospitalizations and deaths. Effective consumer education programs to promote safer food handling practices and other averting behaviors may benefit from consumer awareness of microbial pathogens. This paper investigates US consumers’ awareness of four major microbial pathogens (Salmonella, Campylobacter, Listeria and Escherichia coli) as food safety problems, using a multivariate probit model. The awareness varies among pathogens and the variations appear to be related to differences in the number and severity of illnesses associated with these pathogens. Our findings suggest that awareness of microbial pathogens is associated with food safety perceptions, awareness of potentially risky foods and substances associated with potential food safety hazards, food safety related behaviors, and demographics. There are differentiated effects of variables on awareness of the four pathogens. [Contact: Chung-Tung Lin ]

Consumers' Assessment of the Food Safety Problem for Meals Prepared at Home and Reactions to Food Safety Labeling. 2001. Brian Roe, Mario F. Teisl, Alan S. Levy, Kevin Boyle, Mark L Messonnier, T. Lynn Riggs, Melissa J. Herrmann, and Felicia M. Newman. Journal of Food Products Marketing 6(4):9-26.

To identify if differences in food safety risk can be effectively and credibly communicated, we conducted eight focus groups. This article summarizes these focus groups and reports how consumers frame the issues surrounding the food safety problem and how consumers react to label-based communications of food safety characteristics. We find consumers have broad, moderate food safety concerns, a wide but spotty understanding of food borne illness prevention and consequences, and a healthy skepticism concerning food safety claims. We identity two forms of labeling that show promise with regard to consumer acceptance and credibility in communicating brand-level and package-level differences in the risk of food borne illness and discuss implications for consumer valuation of such differences. [Contact: Amy Lando ]

Foodborne Illness: Perceptions, Experience, and Preventive Behaviors in the United States. 1995. Sara B. Fein, Chung-Tung J. Lin, and Alan S. Levy. Journal of Food Protection 58(12):1405-1411.

Data from national telephone surveys conducted in 1988 and 1993 were used to describe consumer perceptions of foodborne illness. The 1993 data were also used to assess the relationship between the perception that a foodborne illness had recently been experienced and awareness, concern, knowledge, and behavior related to food safety. Respondents described foodborne disease primarily as a minor illness without fever that occurs within a day of eating contaminated food prepared in a restaurant. However, several common pathogens have latency period longer than a day, and experts on foodborne disease estimate that most cases of foodborne illness originate from foods prepared at home. In both surveys, people 18 to 39 years of age were more likely than those in other age groups to believe they had experienced a foodborne illness. In 1993, people with at least some college education were more likely to believe they had experienced foodborne illness than were people with less education. People who believed they had experienced foodborne illness had greater awareness of foodborne microbes and concern about food safety issues, were more likely to eat raw protein foods from animals, and were less likely to practice safe food handling than were those who did not perceive that they had experienced such an illness. [Contact: Chung-Tung Lin ]

Prevalence of Selected Food Consumption and Preparation Behaviors Associated with Increased Risks of Food-borne Disease. 1995. Karl C. Klontz, Babgaleh B. Timbo, Sara Fein, and Alan S. Levy. Journal of Food Protection 58(8):927-930.

Although not well quantified, a portion of food-borne illness results from voluntary behaviors that are entirely avoidable, such as eating raw foods of animal origin or engaging in unsafe food preparation practices. A telephone survey of 1,620 respondents was conducted to assess the prevalence of selected self-reported food consumption and preparation behaviors associated with increased risks of food-borne illness and the demographic characteristics related to such behaviors. The percentages of survey respondents who reported consuming raw foods of animal origin were 53%, raw eggs; 23%, undercooked hamburgers; 17%, raw clams or oysters; and 8%, raw sushi or ceviche. A fourth of the respondents said that after cutting raw meat or chicken, they use the cutting board again without cleaning it. Safer food consumption and preparation behaviors were consistently reported by persons who were female, were at least 40 years old, and had a high school education or less. These findings suggest that risky food consumption and preparation behaviors are common in the United States and that educational campaigns aimed at changing these behaviors may need to be targeted to specific groups of persons. [Contact: Amy Lando ]

Consumer Knowledge of Foodborne Microbial Hazards and Food-Handling Practices. 1995. Sean F. Altekruse, Debra A. Street, Sara B. Fein, and Alan S. Levy. Journal of Food Protection 59(3)287-294.

A national telephone survey was conducted of 1,620 randomly selected U.S. residents who spoke English, were at least 18 years old, and resided in households with kitchen facilities. Respondents were interviewed about their recognition of foodborne pathogens, foods at risk for transmitting infection, knowledge of safe food handling, and food handling practices. One-third of the respondents who prepared meals reported unsafe food hygiene practices: e.g., they did not wash hands or take precautions to prevent cross-contamination from raw meat. Unsafe practices were reported more often by men, persons 18 to 29 years of age and occasional food preparers than by women, persons 30 years old or older, and frequent food preparers. Respondents who identified a food vehicle for Salmonella spp. were more likely to report washing their hands and cleaning cutting boards after preparing raw meat and poultry. The results raise concerns about consumer food-handling practices. The influence of food safety training, food-handling experience, and age on food-handling practices should be studied further. Awareness of a food vehicle for Salmonella spp., for example, may indicate knowledge of the etiology of foodborne disease that promotes safe food handling. Understanding the factors associated with safe food handling will assist in development of effective safe-food instruction programs. [Contact: Amy Lando ]

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings

Preview improvements coming to the PMC website in October 2024. Learn More or Try it out now .

• Advanced Search
• Journal List
• Indian J Med Res
• v.144(3); 2016 Sep

A review on detection methods used for foodborne pathogens

B. priyanka.

1 Department of Applied Zoology, Mangalore University, Mangaluru, India

Rajashekhar K. Patil

Sulatha dwarakanath.

2 Nanoscience Diagnostics, Austin, Texas, USA 78726

Foodborne pathogens have been a cause of a large number of diseases worldwide and more so in developing countries. This has a major economic impact. It is important to contain them, and to do so, early detection is very crucial. Detection and diagnostics relied on culture-based methods to begin with and have developed in the recent past parallel to the developments towards immunological methods such as enzyme-linked immunosorbent assays (ELISA) and molecular biology-based methods such as polymerase chain reaction (PCR). The aim has always been to find a rapid, sensitive, specific and cost-effective method. Ranging from culturing of microbes to the futuristic biosensor technology, the methods have had this common goal. This review summarizes the recent trends and brings together methods that have been developed over the years.

Introduction

Microorganisms, mostly bacteria, are present in gut and skin in human body as normal flora, which are harmless and helpful in many important functions of the body. However, there are many microorganisms including bacteria, fungi and virus that are pathogenic. Gastrointestinal tract is one of the routes through which pathogens enter the human body and cause many foodborne diseases. The foodborne pathogens can enter through contaminated water or contaminated and undercooked food. Hence, it is important to detect the presence of pathogens in food and water before it enters the body to cause a serious outbreak 1 , 2 , 3 . Such organisms mainly include Acinetobacter spp., Bacillus subtilis , B. cereus , Campylobacter jejuni , Citrobacter koseri , C. freundii , Clostridium difficile , C. perfringens , Enterobacter sakazakii , E. cloacae , Escherichia coli O157:H7, Klebsiella oxytoca , K. pneumoniae , Listeria monocytogenes , Salmonella Enteritidis, Salmonella Typhimurium, Shigella sonnei , Staphylococcus aureus , Vibrio cholerae and Yersinia pestis 4 , 5 , 6 , 7 , 8 . The major requirement of detection is in public health, water and food industry, pharmaceutical industry, environment and biodefense 7 , 9 .

Shiga toxin producing E. coli O157:H7 (STEC) has always been one of the major pathogens which are responsible for foodborne outbreaks. The outbreaks can be due to different subtypes of E. coli O157:H7, termed as enterohaemorrhagic E. coli (EHEC) which has got the characteristics of both verotoxigenic E. coli and of a lesser known diarrhoeagenic enteroaggregative E. coli . Contaminated drinking water and water in the swimming pool can also be a cause for E. coli O157:H7 infection. This has been observed in Mangalore, Karnataka, India 10 .

China reported its first outbreak of E. coli O157:H7 in 1986 11 . E. coli O157:H7 has been successfully isolated from humans, livestock and other animals in Fujian, Gansu, Zhejiang, Jiangsu and Anhui 11 , 12 . Powdered infant food (PIF), especially the powdered milk, is prone to pathogenic bacteria. In 2002, powdered milk produced by Wyeth was found to be contaminated with E. sakazakii 7 . Similar outbreak was seen in France where PIF was contaminated with Salmonella sp 6 . In 2010 in Trinidad a study was done with samples taken from 15 farms, and Salmonella sp. was isolated from the farms 13 . Germany saw one of its worst outbreaks in May 2011, when there was an unusually high number of haemolytic-uraemic syndrome (HUS) cases 2 . Turkey is another country that has witnessed a large number of HUS cases as the population of Turkey uses large amount of beef 14 . Mexico, Ireland, Belgium, England, France and Poland have also reported the presence of E. coli O157:H7 in cattle farms, carcass and faeces 14 . STEC detection has been reported from Canada in stool samples screened for viral gastroenteritis 13 . In Tanga region of Tanzania which plays a dominant role in milk marketing, various pathogens have been detected in milk as it offers a perfect medium for growth of microorganisms 15 . Like milk, mozzarella cheese is another consumer product that is prone to get contaminated with L. monocytogenes , E. coli and Pseudomonas fluorescens , and this was seen in Oregon State of USA 16 . Kefir, a fermented milk based beverage, has low percentage of alcohol and is also prone to food contamination by bacteria 4 . L. monocytogenes has been one of the major food pathogens which cause contamination in PIF, which was detected in the USA 17 . L. monocytogenes and L. ivanovii have been found to grow even at 4°C which makes it a major threat as food which is suspected of Listeria contamination has to be tested at the earliest to avoid fatal circumstances. Outbreaks due to some strains of S. aureus such as methicillin resistant S. aureus and Gram positive cocci were detected in food products in China 18 and Spain 19 .

It has been seen that foodborne pathogens can lead to serious outbreaks irrespective of the region. This leads to the spread of disease, more so in infants and aged individuals. Hence, rapid detection becomes important to contain the spread of the pathogen before it leads to a serious outbreak. Various techniques have been evolved to detect the foodborne pathogens. The effort to improve the methods of detection has been a continuous process. The detection methods have been classified into different groups along with their principles, advantages and disadvantages, most of which are discussed in this review. Each method is supported with suitable examples for better understanding of the gradual improvement of the detection systems. The aim is to give an overall gist of the available methods for the detection of the foodborne pathogens.

Culture based methods

Culture based methods have been the oldest methods in detecting the microorganisms, even the pathogenic strains. This method gives a confirmed result regarding the presence of a particular pathogen. The success rate is found to be high, and these methods are cost-effective. However, the biggest drawback in the culture-based method is the slow growth due to which excess time is lapsed to get the final result, which can turn out to be fatal. It must be noted that all these media take up to 18-24 h to give the exact result, indicating the slow turnaround time.

One of the best known examples which shows high success rate and also shows that the method is highly cost-effective is the culture of E. coli O157:H7 on Sorbitol MacConkey agar (SMAC) which is based on the principle of fermentation of sorbitol 20 , 21 . However, the major limitation in this method is slow turnaround time and false positive results due to the emerging serotypes of sorbitol fermenting nonO157 and O157 STEC 20 .

The drawbacks of the SMAC agar can be overcome by the use of chromogenic medium for STEC isolation which has increased specificity and sensitivity. The major advantage of this is the easier discrimination based on colour. Due to the use of the chromogenic substance, the medium is better known as CHROMagar 20 , 22 . Though it is comparatively effective than SMAC, one notable drawback is that CHROMagar is not sensitive to all strains 20 . This was seen in one of the experiments where only one-fifth of the diarrhoeagenic strains were detected when compared to SMAC 20 .

Cefsulodin-Irgasan-Novobiocin (CIN) agar, a selective medium known for better discrimination between bacterial species, was used to differentiate Yersinia enterocolitica and non Y. enterocolitica 23 . Y. enterocolitica chromogenic medium is used where agar has fermentable sugar cellobiose, a chromogenic substrate and selective inhibitor which suppresses the competing bacteria. This indicates that the purple/blue colonies that are formed on the CIN agar are of Y. enterocolitica and Y. pseudotuberculosis which are important food pathogens causing yersiniosis. This method was used to study the contaminated tofu 24 .

Many microorganisms tend to enter starvation mode of metabolism under stress conditions. However, they will remain viable but non culturable (VBNC) which cannot be grown on conventional culture (CC) media, but can signal virulent pathways 25 . Detection of these pathogens is a major challenge for food safety 26 . Since no colonies will be formed, other methods such as fluorescent dyes are used for the detection of VBNC bacteria where different dyes are used. Binding of acridine orange to the VBNC pathogens depends on the ratio of DNA to protein in the cells. Actively reproducing cells appear green whereas slow-growing or non-reproducing cells appear orange. Another dye that is used to detect VBNC is fluorescein isothiocyanate, the principle of which is to detect the enzyme activity of living cells. If there is the presence of any living cells, violet or blue colour is seen 26 . Potable water, pasteurized milk and processed food are vulnerable to VBNC. Some of the foodborne pathogens that fall under VBNC category include C. jejuni , E. aerogenes , E. faecalis , E. coli (including EHEC), Pseudomonas aeruginosa , S. typhimurium , S. dysenteriae , S. sonnei and V. cholerae .

Bacteriophage-derived high-affinity binding molecules (cell wall binding domains, CBDs) have been recently introduced as tools for the detection and differentiation of Listeria in foods as conventional culture (CC) methods are hampered by lengthy enrichment and incubation steps. This when coupled with magnetic separation increases the sensitivity and speed in detection and will be more accurate when compared to the standard diagnostic methods 27 .

Immunoassays

Immunoassays were developed as these were easier to perform, gave faster result and were less expensive. Hence, generally before directly going into polymerase chain reaction (PCR) based methods, immunoassays are performed. Enzyme linked immunosorbent assay (ELISA) is one of the most used immunoassays to date. Antibody purity plays an important role in success of the immunoassays 1 . Along with purity, one more factor that affects the assay is specificity of antibody. Polyclonal antibodies have polyvalency (multiple epitopes to react with). This can affect the reaction, leading to low specificity and sensitivity. It must be noted that there are chances of false positive results. One such result was observed where there was a cross-reaction between E. coli O157:H7, Y. enterocolitica O:9 and Brucella abortus , all the samples obtained from the serum samples of infected cattle 28 .

The use of different substrates in ELISA has a major advantage as the substrates will bind to the respective conjugates specifically and will develop colouration which can be read in an ELISA reader in terms of wavelength. The colour change is visible to the naked eye. However, one of the disadvantages is that the binding of the chemical and conjugate is very specific, and contamination in the intermediate stages can lead to false positive result. One such substrate used is 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) prepared in 0.05 M phosphate-citrate buffer which reacts with bovine serum albumin (BSA) solution 29 . Tetramethylbenzidine is another substrates that is most commonly used in ELISA. It binds to horse radish peroxidase (HRP). The colouration develops gradually. This method was used in development of sandwich ELISA for the detection of Listeria sp 30 . Another most commonly used substrate is p-nitrophenyl phosphate (pNPP) which binds specifically to BSA conjugated alkaline phoshatase 31 . In one of the experiments where detection of E. coli O157:H7 was performed, pNPP was used as the substrate 32 . Bispecific antibodies that recognize human red blood cell (RBC) and the foodborne pathogen L. monocytogenes were engineered. The principle behind this is an initial reduction of a mixture of anti-RBC and anti- Listeria antibodies followed by gradual reoxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process showed red coloured clumps in presence of L. monocytogenes and were readily visible to the naked eyes. This was found to be a simple approach for the rapid and highly specific screening of various pathogens in their biological niches 33 .

From time to time, methodologies in ELISA have been improved to suit the ever emerging new experiments. Blocking ELISA was designed with E. coli O157:H7 LPS as antigen. These were successful in detecting the pathogen in cattle and were found to be more sensitive than the normal ELISA 34 . Indirect ELISA has been used for detecting anti-O157 antibodies in the serum of cattle as well as humans. However, chances of the result being false positive were more due to cross reactivity 34 .

Sandwich ELISA is a modified form of ELISA, in which there will be two antibodies used against one antigen. The sensitivity and specificity is much higher than the existing assays. This kind of ELISA was found to be useful in detecting the Shiga-like toxin ( stx ) in E. coli O157 strains and also non O157 STEC strains and Listeria sp 3 . Polyclonal antibody was used here, with HRP as conjugate for the detection 3 , 30 . The improved version of sandwich ELISA is to detect antibody to the SEF 14 fimbrial antigen (SEF 14 – double antibody sandwich (DAS) - ELISA). This is used for the detection of chicken flocks infected with S. enteritidis . It could discriminate birds infected with S. enteritidis and those infected with Salmonella panama and S. Typhimurium 14 . In another novel experiment of sandwich ELISA assay, detection of stx 2a was performed where the soil samples were spiked with a detection limit between 10 and 100 pg/ml and faecal samples between 100 and 500 pg/ml. When samples were tested by PCR technique, it showed 100 per cent sensitivity and specificity 3 .

The major advantage in reversed passive latex agglutination assay was that 6 h was sufficient for the growth of bacteria, and hence the result obtained was quicker than the culture based 35 . This was tried for determining the toxigenicity of diphtheria toxin of Corynebacterium diphtheriae 35 . Rabbit antitoxin antiserum was used to react with the antiserum with diphtheria toxin.

Monoclonal antibodies are preferred over polyclonal antibody as these have monovalency. In monoclonal antibodies, the antibody is produced against one specific antigen. While sensitivity and specificity are its major positive features, production is a laborious process and is not cost-effective. Various such experiments have been conducted to detect L. monocytogenes , S. Typhimurium , L. innocua and E. coli 36 .

The use of immunoglobulin G (IgG) was the beginning of a new technology, which was useful in targeting virulence in clinical microbiology 37 . However, gradually it is IgY, the counterpart of IgG in chicken egg yolk which has taken over. The major advantages in using IgY is that it is deposited in egg yolk in large quantities and can easily be purified by simple precipitation techniques. This property has made chicken an ideal source for specific monoclonal antibodies. It is very useful in immunotherapy and immunodiagnostics 38 , 39 . This method was proved to be successful in detecting one of the foodborne pathogens C. jejuni when present in low detection limit 40 . A simple and rapid gold-labelled immunosorbent assay (GLISA) has been developed which has the low detection limit of 7.3 log/cfu/g, which is found to be better than many other ELISA methods 40 , 41 . GLISA is commercially available as Singlepath Campylobacter GLISA Rapid Test 40 .

To overcome high detection limits, enrichment steps become important for the detection of pathogens in food products. In the enrichment step, a label-free immunoassay is used that helps in detecting the presence of the pathogen in a much simpler way. A simple and rapid detection is possible through this method with simultaneous enrichment and optical detection. The principle of this method is culture/capture/measure 42 .

DNAzymes are a novel class of molecular probes for detection of bacteria. DNAzymes, also called as DNA enzymes, are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions. These molecules can be isolated from vast random sequence DNA pool by a process called as ‘SELEX’ meaning systematic evolution of ligand by exponential enrichment. This process includes a DNA RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by fluorophore (F) and a quencher (Q). There will be a cleavage where the separation of fluorophore and quencher will lead to increase in fluorescence intensity which makes bacterial detection easy and rapid 43 . Epitope tags which confer specific properties, including affinity for resins or antibodies or detection by fluorescence microscopy, are useful for biochemical and cell biological investigations. This method has been used for the detection of Candida albicans 44 .

Polymerase chain reaction based methods

Kary Mullis in 1985 discovered PCR, which is considered as one of the milestone discoveries in recombinant DNA technology 45 . The principle behind PCR is that genes of various pathogens can be amplified and studied further 46 , 47 . Specific primers are developed for each gene. Agarose gel electrophoresis and subsequent staining with ethidium bromide are used for the identification of PCR products. Since the discovery, various types of PCR have emerged, which are given names according to the changed protocol of the original PCR. In general, the major advantages of PCR are that the process is rapid and sensitive. It is faster than the culture based methods and immunoassays. PCR has now reached new heights where the amplified product can be obtained in just 30 min, and distinguishing between the strains has become much easier as multiple primer pairs are used. The detection limits can become better with time that the DNA amplicon detection limit can be as low as femtograms (10 -15 g) 9 . This can be an alternative to tedious time consuming procedure of culturing and identification of pathogens in food safety laboratories 48 . However, with the advancement in PCR technology, the method has not remained cost-effective although low detection limit will remain the major criteria. PCR technique has developed as a very promising method of detection of the genes in pathogens; however, there are certain disadvantages that make it necessary to develop better methods. The difficulties include cell lysis and nucleic acid extraction, cross-contamination and failed reactions due to the presence of inhibitory substance or competing DNA from the non target cells. This can lead to inconsistent results and reduce the appeal of PCR as a reliable approach 1 . PCR methods are not able to differentiate between the live and dead cells. The primary disadvantage of all the PCR methods is that there are chances of generating false positive signal due to binding to non-specific double-stranded DNA sequences. Therefore, it is extremely important to have well-designed primers that do not amplify non-target sequences.

One of the initial advances in molecular cloning and recombinant DNA technology that revolutionized the detection of foodborne pathogen is the development of a PCR-based technique. In one of the methods, suitable primers were designed based on specific gene fimA of Salmonella and gene afa of pathogenic E. coli for amplification 49 . The size of the amplified product was 120 bp as shown by comparison with marker DNA. This is a rapid, sensitive and reliable technique for the detection of Salmonella and pathogenic E. coli 49 . To design loop-mediated isothermal amplification (LAMP) assays, stx 1, stx 2 and eae genes were chosen as targets 47 . LAMP employs four to six specially designed primers and a strand-displacing Bst DNA polymerase to amplify up to 109 target DNA copies under isothermal conditions (60-65°C) within an hour. The result of LAMP was compared with quantitative PCR (qPCR). The result was obtained within one hour. This method was found to be rapid, specific and sensitive for the detection of STEC strains. One more advantage is the absence of any false positive or false-negative results 47 . During any outbreak, it is important to detect the presence of the pathogen at the earliest. Real-time PCR allows for quantification of the target, and when combined with a rapid cycling platform, results can be generated in 30 min from the start of thermal cycling. Real-time qPCR is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is that it is faster than CC based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms 50 . Ruggedized, advanced pathogen identification device (RAPID) system E. coli O157:H7 kit is a modified version of real time PCR which has the advantage of rapid cycle real-time PCR 51 . An alternate for real time PCR assay is the use of three TaqMan assay sets to detect stx1 , stx2 and rfbE genes. Using multiple PCR assay sets to detect these genes allowed the very specific detection of EHEC from strains which did not possess any of these three genes. The result showed that there was horizontal transfer of stx gene between E. coli strains and in non E. coli enterobacteriaceae strains such as Citrobacter and Enterobacter 52 .

SYBR Green is a cyanine dye which immediately binds to all double-stranded DNA present in the sample. During PCR, DNA polymerase amplifies the target sequence which creates the PCR products. SYBR Green dye then binds to each new copy of double-stranded DNA 53 . As the PCR progresses, more PCR product is generated. SYBR Green dye binds to all double-stranded DNA, so the result is an increase in fluorescence intensity proportioned to the amount of PCR product produced. Real-time PCR has been combined with the dye SYBR Green and was used to detect E. coli strains. The result showed that the presence of SYBR Green increased the discriminating power between the strains 54 . Restriction site specific PCR was performed to detect E. coli O157:H7 which involved the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences. This method does not use endonucleases. It generates amplicons that yield ‘fingerprint’ patterns when resolved on an agarose gel 55 . Multiplex PCR along with SYBR Green was used to detect STEC in O157 and non O157 serotypes of E. coli in cattle faeces 56 . Multiplex PCR uses two sets of primers and two fluorogenic probes for simultaneous and semiautomated detection of Salmonella strains and E. coli O157:H7. This PCR assay was optimized to obtain a strong and reproducible fluorescence signal from probes labelled with two reporter dyes. This helped in immediate and specific detection in meat and faeces 46 , 52 , 57 . Fluorescence was combined with real-time PCR and multiplex PCR for early detection of stx1 , hly and eae genes. This led to a billion fold amplification when experiment was performed under isothermal condition 58 , 59 . Large-scale multiplex (LSplex) uses 800 specific primer pairs. It can successfully amplify different pathogens whether it is Gram positive or Gram negative. It generated stronger signals with just 10 ng of DNA as compared to the ones which used 2-5 µg of DNA 9 . One aspect that can be improved in LSplex PCR is that its detection limit can be reduced to pico (10 −12 g) or to femtograms (10 −15 g). This will be very desirable in detection of every clinical, food or environmental samples 9 . Fluorescent amplification-based hybridization PCR shows good results in fluorescence intensity which is the most important aspect in detection of pathogens. It is found that fluorescent signal for E. coli O157:H7 was 6.40 while that for other related pathogens was 2.50 59 . It is also cost-effective. Reverse transcription PCR (RT-PCR) is another technique which uses reverse transcriptase enzyme to produce DNA from RNA followed by the normal PCR technique. This technique is used to detect virus causing dengue 37 . Detection of Salmonella sp. using real-time PCR is also reported in pork chop and sausage samples using SYBR Green dye in RT-PCR 60 , 61 . Real time RT-PCR has shown great potential for detecting viable pathogens such as S. enterica where mRNA is detected. In one of the studies, expression of Salmonella specific sigDE operon which encodes invasion proteins was studied and it was found that the sigDE could be a useful viable marker for the bacteria 62 .

The use of reporter quencher technique has been known since the early 1990s, which has developed over time 63 . The nucleic acid amplification technique is an indispensible tool in clinical diagnostics. Accurate and specific quantification of pathogen is very important. Hence, a new mediator probe has been developed which works on the reporter quencher methodology where release of mediator triggers signal generation of a complementary fluorogenic reporter probe. This technology was applied to detect and amplify S. aureus and E. coli 64 . Novel nucleic acid probes known as molecular beacons have been developed allowing for the rapid and specific detection of disease. Molecular beacons are hairpin-forming oligonucleotides labelled at one end with a quencher and at the other end with a fluorescent reporter dye 65 .

Conventional pathogen detection methods, such as microbiological and biochemical identification, are time-consuming and laborious while immunological or nucleic acid-based techniques require extensive sample preparation and are not amenable to miniaturization for on-site detection. Novel biological recognition elements are studied to improve the selectivity and facilitate integration on the transduction platform for sensitive detection. However, the probe that is designed has to be very specific. Bacteriophages are one such unique biological entity that show excellent host selectivity and have been actively used as recognition probes for pathogen detection 66 . When there is a necessity of differentiating pathogens, for example, E. coli and other enteric bacteria, gene gadAB present in E. coli strains can be obtained from the consumer food materials. However, Shigella is the only species which is gadAB -positive. To overcome the false positive results, gadAB gene can be used as a marker for just E. coli . This indicated that gadAB marker was suitable as pre-screening marker for E. coli 67 . This led to a large-scale genome comparison. This method is called octamer-based genome scanning 68 , 69 . The markers where gene is used are called as DNA probes. Protein probes such as green fluorescent protein (GFP) obtained from gfp gene is known for its fluorescence. This gene is obtained from the jellyfish Aequorea victoria . Expression of selectively inducible gfp gene in a plasmid transformed strain of E. coli O157:H7 was found to be a useful tool in the detection of the pathogen 70 . GFP protein produced by gfp gene shows a characteristic emission peak at 509 nm which indicates the presence of the organism 70 .

Biosensors are the latest among all the detection systems, some of which have better detection limits which significantly reduce and also eliminate the drawbacks associated with PCR techniques 1 , 66 , 71 , 72 , 73 , 74 , 75 , 76 . Biosensors are the devices for pathogen detection that generally consist of three elements, which are a biological capture molecule (probes and antibodies), a method for converting capture molecule – target interactions into a signal and an output data 2 . Despite better detection efficiencies, results derived using molecular biology methods can be affected by the various food matrices. One of such detection studies was done on Y. enterocolitica , a pathogen that can cause yersiniosis in humans and animals 77 . Improvements in sample preparation, data analysis and testing procedures, molecular detection techniques can simplify and increase the speed of detection. The major advantage of the biosensors is that these can detect the pathogens at low detection limits with high specificity and sensitivity, but the biosensors will require highly specific and expensive instruments, with compatible computer software, to give accurate results. Hence, these methods may not be always cost-effective.

Electrochemiluminescent assays are performed in 96-well plates and are based on electrochemical stimulation of reporter molecules such as ruthenium (II) trisbipyridal (Ru(bpy) 3 ) 2+ chloride which are attached to antibodies. The detection in this method is at a low concentration. A slightly improved version of this is called cytometric bead assay which uses a fluidic approach and have red and infrared fluorophores. These give out orange fluorescence when exposed to the electrode 1 . A lab-on-a-chip integrates cell pre-concentration, purification, PCR and capillary electrophoretic (CE) analysis. It is a microdevice which has a 100 nl PCR reactor and 5 cm long CE column for amplicon separation. Detection limit is 0.2 cfu/µl 78 . It can be used in detection of E. coli K12. Similar to lab-on-a-chip assay is cell-phone based on E. coli detection platform for screening of liquid samples. Battery powered inexpensive light emitting diodes are used. Excitation of sample is done, and the emission from the quantum dots is imaged using a phone camera unit. It was demonstrated for fat free milk mainly to detect Salmonella sp. where the detection limit of 5 to 10 cfu/ml was achieved 78 . Similarly, an exposure to antibody-quantum dot conjugates was used to detect E. coli and S. Typhimurium 79 . CdSe/ZnS quantum dots exhibited fluorescence emission shift when conjugated to antibody or DNA aptamers that are bound to bacteria. This shift in emission peak occurs when the quantum dots encounter the bacterial surface 80 .

A surface plasmon resonance (SPR) immunosensor was designed by means of a subtractive inhibition assay using goat polyclonal antibodies for E. coli O157:H7. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 74 . Fluorescence resonance energy transfer (FRET) utilizes an antibody recognizing cell surface epitopes of the target cell. It makes use of complementary oligonucleotides that are modified with fluorochromes. Fluorescence is detected using the sensors. E. coli O157:H7 and Salmonella were detected using FRET. The advantage of FRET is that it is simple, fast by giving result within five minutes. It is inexpensive and highly sensitive 81 .

Optical biosensors have been proven to have better detection system and separation of pathogens. These biosensors include optical fibres, planar wave guides, SPR and microarrays. Their compact design and label-free detection lead to specific and sensitive detection and this is a major advantage of optical biosensors 75 .

Nanobiotechnology is the latest approach for detection of pathogens. Aptamers are attracting an increasing amount of interest in the development of sensors for proteins, DNA and small molecules. An experiment design based on the combination of nucleic acid aptamer with polydiacetylene showed 98.5 per cent detection of E. coli O157:H7 (203 clinical faecal samples) when compared with the standard culture 72 . High affinity and specificity are found in aptamers. Gold nanoparticles (GNPs), silver nanoparticles and bioconjugated nanoparticles which give fluorescence have been used in aptamers 76 , 82 , 83 . GNPs have electronic, photonic and catalytic properties making their applications unique. GNPs can be used in colorimetric methods due to their optical properties 76 . These are non toxic and can easily conjugate to antibodies 82 . A bioconjugated, nanoparticle-based bioassay provides a high fluorescent signal for bioanalysis. An attempt was done using E. coli O157 cells in beef sample in a 384-well microplate format 83 . A new approach involves physical damage to the bacteria using a combination of pulsed laser energy and absorbing nanoparticles. When irradiated, nanoparticles absorb energy and when relaxed give out heat, which damages the cells. GNPs have been used for this method 82 .

Other detection methods

DNA microarray is gaining importance currently and has become a useful tool due to its rapidness, sensitivity and specificity and it allows high throughput analysis. Various studies have been conducted to detect waterborne pathogens, marine fish pathogens, which indirectly will be a threat to humans due to fish consumption 6 , 9 , 15 . Li et al 58 reported the detection of foodborne pathogen microarrays designed to target internal transcribed spacer (ITS) sequences. In one of the studies, 10 pathogens were tested for their presence in PIF 6 . B. cereus , E. coli , L. monocytogenes , P. aeruginosa , S. enterica , S. aureus , V. parahaemolyticus and, C. freundii were detected using this method. E. sakazakii , K. pneumoniae , K. oxytoca , Serratia marcescens and A. baumannii are associated with contaminating PIF 6 . DNA microarray technique is used to detect these pathogens. ITS regions of five Bacillus sp. B. anthracis , B. cereus , B. thuringiensis , B. mycoides and B. weihenstephanensis were examined as these possess a high homology at DNA level, making it difficult to differentiate. DNA microarray was the solution to this problem 6 . PulseNet, a national molecular subtyping network for foodborne disease surveillance is playing a key role in detecting each of the outbreaks by the pathogens 5 . It mainly helps in reducing product recalls, restaurant closures and related mechanisms after the outbreak. This is done in local, State and public health and regulatory agency laboratories 5 .

Ultrafiltration, immunomagnetic assays (IMS), immunochromatic assay (ICA), flow cytometry (FC) and lyophilization are some of the conventional methods. Ultrafiltration has been recognized as an effective procedure for concentration and recovering microbes from large volumes of water and treated waste water 52 . Conventional IMS procedure uses an external source to capture magnetic particles against the side of the test tube which leads to poor results due to high background microflora 84 . Hence, PickPen IMS is used which increases the throughput compared to the conventional IMS. The difference is that there is an intrasolution magnetic particle transfer device in PickPen IMS which detects E. coli O157:H7, Salmonella sp. and L. monocytogenes that are prevalent in various samples. Its consistent recovery of immunobeads has high throughput and lower carryover of background microflora 84 , 85 . In one of the experiments in detecting S. Typhimurium , IMS was combined with CC, with PCR and with Fourier transform infrared spectroscopy (FTIR) as IMS-CC, IMS-PCR and IMS-FTIR where combination of IMS with FTIR was found to be the most accurate and rapid test 76 , 86 .

FC is a sensitive analytical technique which can rapidly monitor physical states of bacteria. Fluorescent probes are used to detect E. coli O157:H7, P. aeruginosa , P. syringae , S. Typhimurium and Cyclospora cayetanensis (in oocytes) 75 , 87 .

ICA has been a useful, simple, rapid, highly sensitive, specific method and does not require expensive equipment or reagents. It can be judged by naked eye in terms of cfu/ml. Immunomagnetic nanoparticles use nanopure iron as core coated with E. coli O157:H7 polyclonal antibodies in combination with ICA 11 . It has been found that lyophilization prior to direct DNA extraction from bovine faeces improves the quantification of C. jejuni 88 .

An ideal detection method needs to satisfy five premier requirements – high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and use of specialized equipment) and cost effectiveness. For example, culture takes long time to give the results. On the other hand, PCR, antibody-based techniques and biosensors offer shorter waiting time, but these require the use of expensive reagents and sophisticated equipment which make the method expensive.

In this review, various methods of detection of pathogens which have been developed and improved from time to time have been discussed with the pros and cons of the respective methods ( Figure ). An important point that needs to be stressed here is that the search for better detection methods of pathogens cannot be stopped at one point. This will be an area of research and newer experiment will be evolving to make the detection systems rapid, sensitive, specific and cost-effective to the maximum extent.

Schematic representation of the methods for the detection of pathogens. ELISA, enzyme linked immuno sorbent assay; DAS, double antibody sandwich; PCR, polymerase chain reaction; LAMP, loop mediated isothermal amplification; RSS, restriction site specific; RT, real time; ECL, electrochemilumenescence; FRET, fluorescence resonance energy transfer; IMS, immunomagnetic assay; ICA, immunochromatic assay; FTIR, fourier transform infrared spectroscopy.

Conflicts of Interest: None.