Polymerase Chain Reaction (PCR) based procedures work on the fact that given species of pathogens posses 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. The purpose of this paper is to summarize the various PCR diagnostics approaches of detecting pathogens in food (Bruns 2007).
PCR procedure refers to a scientific technique that enables replication of single strands of genetic mark-up i.e. DNA to the desired copies and sequence. Quantitative PCR commonly referred to as Real time PCR has made it possible to quantify RNA or DNA present in a given sample. However, for 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 as Reverse Transcriptase Polymerase Chain Reaction; RT-PCR. Due to its high specificity and sensitivity, PCR has gained wide-spread usage as a diagnostic tool in various settings.
Hocking (2006) states the use of polymerase chain reaction in the invitro amplification of a unique nucleic acid sequence to a given pathogen permits robust diagnosis with increased specificity and sensitivity. He also points out that PCR procedures are applicable to practically any harmful bacteria and is primarily employed in conjunction with pulsed-field gel electrophoresis in the detection of bacterial pathogens in food. There are three steps that are involved in DNA replication during PCR procedure. Step 1 of this process involves three important stages which are denaturation, annealing and extension. The table below shows the standard RT-PCR protocol for 50 µl reactions done by Schanke in a study that sought to detect the BCR/ABL transcripts (Schanke 2006).
|DNA Polymerase Protocol and Cycling Conditions for One-Step RT-PCR.|
|Reaction (50 µl total reaction volume):||Final Concentration:|
|H2O||X µl||to 50 µl|
|20X PCR Buffer||2.5 µl||1X|
|25 mM MgCl2||3 µl||1.5 mM*|
|2.5 mM dNTP mix||4 µl||200 µM each nucleotide*|
|10X MasterAmp PCR Enhancer||5 µl||1X*|
|20 µM Primer 1||2.5 µl||1 µM|
|20 µM Primer 2||2.5 µl||1 µM|
|RNA Template||X µl||0.1 – 1 µg*|
|25 mM MnSO4||1 µl||0.50 mM|
|TthDNA Polymerase, 5 U/µl||1 µl||5 units|
|First Strand Synthesis:||60°-65°C*||20-45 minutes|
|Then 30 cycles of :|
|Extend:||65°-72°C**||1 minute per kb product|
Application of PCR Technique
According to Pattrinos & Ansorge (2005) Real-time PCR meets the accurate and robust detection of disease-causing bacteria in food samples which is essential in tracing of 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) points out that one of the greatest problems facing PCR based diagnosis of food pathogens is to differentiate between dead and living 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 diagnosis 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) points out numerous protocols have been developed and validated to be applicable in the detection of Salmonella, Listeria and Escherichia coli in environments and in food samples.
Limitations of PCR Procedure
According to Bruns et al. (2007), despite its high specificity and sensitivity, polymerase chain reaction results from one laboratory are difficult to replicate in other laboratories due to use of complex equipment, sensitive reagents and the need for qualified personnel. Therefore, there is need for proper validation on the basis of consensus criteria, which is a vital prerequisite for any successful diagnostic procedure that is 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 the five core pathogens; enterohemorrhagic Eschericia coli, Salmonella enteric, Listeria monocytogenes, thermophilic Campylobacter spp and Yersinia enterocolitica (Hui 2006). Despite these limitations, PCR remains the most sensitive, robust and specific method for pathogen detection in food substances.
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.
Schanke, J. 2006. A One-Step MasterAmp: RT-PCR Procedure to Detect the BCR/ABL Transcript of the Philadelphia Chromosome and the Enterovirus Genome. Epicentre Biotechnologies, 4.2: pp. 67-89.
Zourob, et al. 2008. Principles of bacterial detection: biosensors, recognition receptors and Microsystems. New York, NY: Springer Science.