PCR technologies for the detection of pathogens in the food industry

Food safety is critically important to the public health of the consumer and the economic sustainability of the agri-food sector. The consumer wants assurance that food is safe and for the food industry the economic implications and loss of goodwill associated with a food poisoning incident or scare has increased the necessity to provide assurance on food safety.

To provide assurance of safety, foods are tested to ensure they conform to micro – biological criteria for particular microbial pathogens and / or hygiene indicator organisms. There are many challenges in the detection of food pathogens; in particular, they are generally present in very low numbers in the food (often < 100 cfu g-1) in the presence of up to one million other microbial flora. The pathogens may be attached to the food surface or imbedded in a complex food matrix. The microorganisms on the food are often in an injured condition as result of storage at chill, frozen temperatures, low pH or aw or the presence of food preservatives. These injured organisms can be difficult to recover but as they do have the potential to cause illness, their detection is of public health importance.

Traditional methods for the detection of bacterial pathogens from foods rely on culturing of the organisms on agar plates (one to two days) and usually necessitates an initial liquid enrichment step (one to two days) to increase the numbers of the target organism, and to enable recovery of injured pathogens and their detection from the food. The suspect colony on the agar plate can then be identified by morphological, immunological or biochemical means. These traditional culture based methods are time consuming, taking five to seven days to detect specific pathogenic micro-organisms. However, while more rapid alternative methods are needed and are becoming more widely available, all microbial criteria are still based on culture results (CFU/G) and thus any alternative methods used by the food industry must give a result which shows equivalence with standard cultural method on validation. The process to show equivalence of rapid / alternative qualitative or quantitative method is outlined in “Microbiology of food and animal feeding stuffs: protocol for the validation of alternative methods” (ISO 16140).

Additionally if a sample is positive for a particular pathogen, it may be for public health and legal (product withdrawal) essential to have a cultured isolate which can be fully characterised and tracked. Therefore, an ideal alternative method for the food industry is one that can reliably and rapidly screen out negative samples but is compatible with standard culture method to facilitate cultural isolation from positive samples. The general approach for use of rapid methods to detect pathogens in foods is outlined in Figure 1.

Approaches to rapid detection of pathogens have been developed based on one of two approaches, immunological or molecular methods. Of these, molecular based approaches have great potential as ongoing advances in the level of genomic information available for foodborne pathogens are exploited to develop methods to detect and characterise microorganisms.

Molecular diagnostic techniques are based on the detection of a fragment of genetic material (nucleic acids i.e. DNA or RNA) which is unique to the target organism. The level of specificity is based on target gene used and thus may be at the level of bacterial genera, species, serogroup or virulence or phenotype related. Several targets can be included in a single multiplex assay. Methods which include an amplification step for the target DNA/RNA are now routine in molecular diagnostic assays. These methods increase the target nucleic acid material by up to a million fold and are particularly important in the arena of food microbiology where one of the major hurdles as described above is the recovery and detection of very low numbers of a particular pathogen. However, even with the incorporation of an amplification step, a liquid enrichment of the food sample (24 to 48 hours) is generally still required to yield sufficient nucleic acid material from the target organism for the reaction.

Polymerase Chain Reaction

The most popular method of amplification is the polymerase chain reaction (PCR) technique. In PCR, a nucleic acid target (DNA) is extracted from the cell and denatured into single stranded nucleic acid. An oligonucleotide primer pair specific for the selected gene target, along with an enzyme (Taq polymerase, a thermostable and thermoactive enzyme originally derived from Thermus aquaticus) in the presence of free deoxynucleoside triphosphates (dNTPs) is used to amplify the gene target exponentially resulting in a double replication of the starting target material.

 In conventional PCR this reaction is carried out in a programmable block heater called a thermocycler which provides the necessary controlled thermal conditions needed to achieve amplification. The amplified PCR products are then separated by gel electrophoresis, stained with ethidium bromide and visualised using ultraviolet light. (Photo 1). This type of PCR can be used for the identification of specific groups of pathogenic bacteria.

A major concern when applying PCR for the detection of pathogens in foods is the possible reporting of false negative results as a result of interference with target-cell lysis during nucleic acid extraction, nucleic acid degradation or direct inhibition of the PCR. Therefore, both internal and external controls should be included with each assay to monitor assay performance. External controls monitor the use of instrumentation, the assay reagents and ensure no DNA cross-contamination has occurred. Internal amplification controls (IAC) involve the use of a separate PCR assay that are included in the same tube or well as the detection assay. The IAC amplifies a nucleic acid such as an exogenous target sequence and monitors the efficiency of each reaction, providing assurance that amplification and detection are working effectively. The International Organisation for Standardisation (ISO) has developed ISO 22174 “Microbiology of food and animal feeding stuffs- polymerase chin reaction for the detection of food borne pathogens – general requirements and definitions” in which principal criteria and parameters for PCR performance as a diagnostic tools are defined. Table 1 outlines gene targets used for a range of food pathogens in developed PCR tests.

Real time PCR

A disadvantage of conventional PCR is that it is labour intensive and thus is slow if large numbers of samples are to be processed and an automated system called real-time PCR is now increasingly replacing the conventional protocol. Real-time PCR allows continuous monitoring of the amplification process through the use of fluorescent double stranded DNA intercalating dyes or sequence specific probes. The amount of fluorescence after each amplification cycle can be measured and visualised in real time on a computer monitor attached to the real time PCR machine. A number of dye chemistries have been reported for use in the method including Sybr green I intercalating dyes, hybridisation probes (HybProbes), dual-labelled oligoprobes (TaqMan probes) and hairpin oligonucleotides (molecular beacons). In all cases, a fluorescent signal is generated during the PCR process that is captured by one of the several different commercial real-time instruments. Regardless of the signal chemistry used, real-time PCR not only allows quick determination of the presence / absence of a particular target, but can also be used for the quantification of a target which may then be related to microbial counts.

Real-time PCR is considerably faster than conventional PCR, less prone to operator error and more convenient as the PCR amplification and detection are all carried out in one machine. The use of a closed system for amplification and detection minimises the potential for amplicon carryover contamination. Although expensive in capital terms, real-time PCR allows the processing of large number of samples with minimal labour.

Reverse transcriptase polymerase chain reaction (RT-PCR)

While DNA is generally selected as a target molecule in designing PCR assays for food pathogens, a limitation of this approach is that it is not possible to distinguish between viable and non-viable bacteria, though this is somewhat overcome by sample enrichment which increases the numbers of viable cells and target DNA. mRNA which has a short half life, is a better target for the determination of viability. Reverse transcriptase polymerase chain reaction (RT-PCR) is a variation of the PCR reaction and employs the enzyme reverse transcriptase to convert messenger RNA (mRNA) into complementary DNA (cDNA) which is subsequently amplified by DNA PCR allowing an exponential increase in the amount of mRNA in the form of cDNA copies. One of the main difficulties with this technique is that isolation of RNA is technically more difficult than DNA and is also less stable. RT-PCR has been used to monitor cell viability in bacteria of relevance for the food industry, such as Salmonella and Listeria monocytogenes and is particularly suited to detection of viruses and parasites as they cannot be cultured.

Commercial PCR kits

There are a number of manufacturers now producing PCR kits for detection of food pathogens. This includes a kit produced by BAX® (Dupont Qualicon) which is based on a conventional PCR approach with detection of amplified target by gel electrophoresis for the detection of Listeria and Salmonella. A real time PCR kit system for Salmonella is available from Perkin Elmer Taqman® (Roche Molecular systems).

Conclusion

It is clear that the PCR is a tool which if used with appropriate quality controls and is fully validated against the culture methods (ISO 22174, ISO 16140) is now a diagnostic tool which can be used by the food industry to give rapid and sensitive assessment of the microbial profile of a food sample. The key to better uptake of this technology in the food industry is continued focus and efforts on international validation and standardisation of PCR assays for food pathogens. Initial capital cost and higher running costs are a consideration but they may be off set by savings from obtaining results earlier. PCR can play a key role in risk based food safety management systems.

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About the Author

Dr Geraldine Duffy is Head of the Food Safety Department at Teagasc, Food Research Centre, Ireland. Her research focuses on the transmission and control of microbial pathogens in the farm to fork food chain and she has published over 80 peer reviewed publications in this area. She has been an active participation in EU Framework research programmes for over 10 years and is co-ordinator of the integrated project Prosafebeef (www.prosafebeef.eu)