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.
Table 1 outlines gene targets used for a range of food pathogens in developed PCR tests. 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.
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.
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.
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.
There are a number of manufacturers now producing PCR kits for detection of food pathogens. 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 , ISO 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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Chiu, T. International Journal of Food Microbiology 1; 97 3 Ellingson, J. Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to-eat meat products. Molecular and Cellular Probes. Jin, U. PCR method based on the ogdH gene for the detection of Salmonella spp.
PCR Methods in Foods
Journal of Food Protection 66 11 , Jung, Y. Polymerase chain reaction detection of Listeria monocytogenes on frankfurters using oligonucleotide primers targeting the genes encoding internalin AB. Journal of Food Protection 66 2 Koo, K. Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set.
Applied and Environmental microbiology. Krause, M. Comparative, collaborative, and on-site validation of a TaqMan PCR method as a tool for certified production of fresh, campylobacter-free chickens. Applied and Environmental Microbiology, 72, Longhi, C. Detection of Listeria monocytogenes in Italian-style soft cheeses. Journal of Applied Microbiology 94 5 , Lunge, V. Journal of Microbiological Methods 51, Malorny, B. Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples.
Journal of Association of Analytical Communities International 87 4 Evaluation of a PCR assay for the detection and identification of Campylobacter jejuni and Campylobacter coli in retail poultry products. Research Microbiology 4 McCabe, E. J Microbiol Methods.
New PDF release: PCR Methods in Foods (Food Microbiology and Food Safety)
O Grady, J. Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssr A gene, a novel diagnostic target. Food Microbiology, 25, Rapid Detection and Quantification of E. Journal of Applied Microbiology Oyofo, BA. Rapid and sensitive detection of Campylobacter spp. Paton, A. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohaemorrhagic E.
Perelle, S. International Journal of Food Microbiology, , The level of system dedication to the food industry, such as the DuPont Qualicon BAX, is sometimes a clue to good software. One needs to consider whether lyophilization benefits the user or the manufacturer. Assuming adequate shelf life, use of a liquid product can mean one less preparation step.
Look for UNG-containing kits that do not require any intervention steps on the part of the user. Alternatively, the same capability for double duty may be wasteful if never used.
A minute denaturation of the DNA molecule at the end of amplification and detection allows sub-species analyses to be conducted. This is by no means comparable to serotyping, but it does give new information, previously unavailable in PCR The more direct form of FRET may produce more distinctive melting curves. A quick look at melting curves of the different systems shows the differences. Both work well in foodborne pathogen analysis. Application support and system support are critical. Speaking with a knowledgeable or unknowledgeable company representative in a meeting, trade show or workshop can be one sign of the future in working with that company.
These include Salmonella spp. It should be noted that with regard to the latter two microorganisms, there is a gray area in the PCR industry; specifically, the detection of H7 or the detection of H7 within O It is worthwhile for potential users to ask the PCR company if the test wanted is actually the test offered by the system. Additionally, PCR tests can detect spoilage organisms in beer and can qualitatively and quantitatively detect genetically modified organisms GMOs in soy and maize, as well as used for general GMO screening of other food types.
GMOs can be analyzed qualitatively and quantitatively.
Encyclopedia of Food Microbiology - 2nd Edition
First, the speed to result is the reality of a two-day pathogen test, consisting of 20 to 24 hours of enrichment and then an hour or two later having a result. Second, PCR technology offers flexibility in terms of the detection of different pathogens within a run, as well as the detection of multiple organisms within a test depending on the test.
Third, it offers security of result in that PCR is a confirmatory test. Most food testing applications employ a method known as real-time PCR detection — combining the amplification and detection stages of the process so that amplification is monitored continuously during the exponential phase. Real-time detection is more accurate and the result can also be quantified.
These fluoresce only when bound to double-stranded DNA and the increase in fluorescence can be measured at each cycle. This makes it difficult to quantify the result accurately. The problem can be overcome to some extent, but only by adding an extra stage at the end of the PCR process. A second, more accurate and reliable method is to use fluorescent reporter probes.
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This method utilises an additional primer, the probe, which also binds specifically to the target DNA sequence during annealing. Probes have a fluorescent reporter dye at one end and a quencher dye, which inhibits fluorescence, at the other. During the extension stage the probe is broken apart by the DNA-polymerase and begins to fluoresce more strongly.