Assurance of food safety and quality are the most important aspects of global food production, consumption and trade because of increased consumer awareness towards quality and safety of global food products. For the recognition and prevention of problems to ensure quality and safety, the detection of food borne pathogen is of utmost importance at all levels of food production to build the consumer confidence. For several decades, many food microbiologists have targeted their research goals at the development of rapid methodology for the detection of food pathogens.
With these dedicated efforts, food microbiology laboratories now have a good array of detection methods and automatic technologies like enzyme immunoassay, Polymerase Chain Reaction (PCR), and microarrays which may cut test times considerably. A
mongst these, nucleic acid amplification strategies (PCR) and advanced detection methodologies (Real Time PCR) have been key factors in the progress of molecular microbiology and have been used widely for the detection of food borne pathogens like toxin producing Escherichia coli (E. coli O157:H7), Listeria monocytogene, Salmonella sp., Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter jejuni, Enterobacter sakazakii and Staphyloccus aureus.
PCR has revolutionized traditional microbiological analysis by allowing detection of pathogenic micro-organisms directly in the food, without the necessity of classical isolation and identification. Since its discovery it has become an essential analytical tool for researchers working in the foodborne pathogens field.
Multiplex PCR offers a more rapid detection as compared to simple PCR through the simultaneous amplification of multiple gene targets. The essential principle of mPCR is analogous to standard PCR. However, several sets of specific primers are utilized in mPCR assay whereas just one set of specific primers are utilized in conventional PCR assay.
However, the most used weapon for the rapid detection of food borne pathogens is Real Time PCR (RT-PCR), also referred to as quantitative PCR or q-PCR. RT- PCR or q- PCR is different from simple PCR. Reactions are characterized by the purpose in time during cycling when amplification of a PCR product is first detected instead of the quantity of PCR product accumulated after a hard and fast number of cycles.
RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations.
At present, there are a number of different companies that are manufacturing PCR kits for detection of food pathogens. Kits which are based on conventional PCR approach are being produced by BAX (Dupont Qualicon), Promega Corporation, QIAGEN. These brands have designed the kits for quickly and accurately to detect foodborne pathogens in a broad range of foods and associated samples. iQ-Check from Bio-Rad, DuPont Qualicon BAX, foodproof from BIOTECON Diagnostics, AnDiaTec from Roche, MicroSEQ from Applied Biosystems, and SureTect from Thermo Scientific are the some of the RT-PCR kits available in the market.
RT-PCR, with continued improvements in instrumentation and chemistry, is becoming a ubiquitous method in the analysis of food, to detect pathogens, such as viruses or bacteria, to identify allergens in food, and to detect what species of plants or animals are in a food, and in what proportion, with high specificity. A range of fluorescent probe chemistries are now available and nanoparticles are opening up new opportunities in RT-PCR, owing to their higher sensitivity and short detection times.
However, there are many challenges yet to be addressed. Recent progress in RT-PCR analyses, such as microfluidic integrations, presents a positive outlook for gene-based point-of-care food analysis at a much lower cost in the near future. The development of microfluidics-based quantitative RT-PCR for molecular diagnosis is becoming a burgeoning field of research.
Manisha Mathur, Research fellow, Advanced Milk Testing Research Laboratory, Post Graduate Institute of Veterinary Education and Research (PGIVER), Jaipur