Polymerase Chain Reaction (PCR) based procedures work on the fact that given species of pathogens possess an inimitable RNA or DNA sequence that can be employed for its identification. PCR has made it possible to generate enormous quantities of a specific DNA sequence from a multifarious assortment of heterogeneous sequences. After the amplification of the desired DNA sequence into million copies, the sequence can be identified through other techniques such as DNA hybridization, whereby the desired sequence is hybridized with a probe and gel electrophoresis. Quantitative PCR commonly referred to as Real-time PCR has made it possible to quantify RNA or DNA present in a given sample.
However, retroviruses (viruses whose genomes are composed of RNA as a substitute of DNA) such as influenza viruses are identified through the generation of copies of complementary DNA commonly denoted as cDNA from their RNA copies using reverse transcriptase. The generated complementary DNA copies are then amplified by polymerase Chain Reaction Procedures. This procedure is commonly referred to as Reverse Transcriptase Polymerase Chain Reaction; RT-PCR.
Due to the high specificity and sensitivity associated with PCR, has gained widespread usage as a diagnostic tool in various settings. Nevertheless, the primary aim of this paper is to provide a literature review on the use of PCR diagnostic tools in the detection of pathogens if food (Bruns, 2007).
Hocking (2006) states that the use of polymerase chain reaction in the in vitro amplification of a nucleic acid sequence unique to a given pathogen permits robust diagnosis with increased specificity and sensitivity. He also points out that PCR procedures apply to practically any harmful bacteria and are primarily employed in conjunction with pulsed-field gel electrophoresis in the detection of bacterial pathogens in food.
According to Pattrinos & Ansorge (2005), Real-time PCR meets the accurate and robust detection of disease-causing bacteria in food samples essential for tracing outbreaks of bacterial pathogens within a specific food supply chain and food quality assurance. He points out that Real-time PCR-based analysis meets the above requirements since it highly reduces analysis time relative to conventional serological and biochemical identification procedures. Zourob, et al. (2008) point out that one of the greatest problems facing PCR-based diagnosis of food pathogens is to differentiate between death and life pathogenic cells. To overcome these problems, researchers developed mRNA-based real-time PCR assays instead of DNA-based assays. The former has the advantage of indicating the viability of the pathogen.
As stated in Maurer (2006) the development of polymerase chain reaction has overcome the need for high quantities of organisms nearly 105 cells/ml or more required for identification by the early DNA procedures. According to Sachse & Frey (2003) polymerase, chain reaction procedure has been commercialized for the detection of trace quantities (approximately 100 cells/ml in 4 hours) of Legionella and Salmonella. Bisen et al (2010) point out numerous protocols have been developed validated for employed in the detection of Salmonella, Listeria and Escherichia coli in environmental and food samples.
According to Bruns et al. (2007), despite it is high specificity and sensitivity, polymerase chain reaction results from one laboratory are difficult to reproduce by other laboratories due to use of complex equipment, sensitive reagents and need for qualified personnel. Hence, there is a need for proper validation on the basis of consensus criteria, which is an utter prerequisite for any successful employed of diagnostic procedures based on PCR.
For Hui (2006) this was the basis for the approval of the FOOD-PCR project by the European Commission. The fundamental aim of this project was standardization and validation of diagnostic use of polymerase chain reaction in the detection of pathogenic bacteria in food materials, specifically five core pathogens: enterohemorrhagic Eschericia coli, Salmonella enteric, Listeria monocytogenes, thermophilic Campylobacter spp, Yersinia enterocolitica. Despite these limitations, PCR remains the most sensitive, robust and specific method for pathogen detection in food substances.
References
Bisen, P. S. et al. 2010. Molecular Diagnostics: Promises and possibilities. New York, NY: Springer Science.
Bruns, D. E. et al. 2007. Fundamental of molecular diagnostics. Philadelphia, PA: Saunders Elsevier.
Hocking, A. D. 2006. Advances in food mycology. New York, NY: Springer Science.
Hui, Y. H. 2006. Food Biochemistry and food processing. Malden, MA: Wiley-Blackwell.
Pattrinos, G. P., & Ansorge, W. 2005. Molecular Diagnostics. Oxford: Elsevier Academic Press.
Maurer, J.2006. PCR methods in foods. New York, NY: Springer Science.
Sachse, K., & Frey, J.2003. PCR detection of microbial pathogens. Totowa, NJ: Human Press.
Zourob, et al. 2008. Principles of bacterial detection: biosensors, recognition receptors and Microsystems. New York, NY: Springer Science.