Food safety assays using quantitative polymerase chain reaction
Conventional food assays used to detect bacterial contamination such as protein-based immunoassays, ELISA, or others are not only time consuming but costly, less accurate and labor intensive as well.
Polymerase chain reaction (RT-PCR), also known as quantitative polymerase chain reaction (qPCR), is a rapid and cost-effective quantitative method for detecting the presence of target DNA segments in samples and assisting in the identification contamination of food by biological contaminants.
Since PCR is capable of amplifying a specific fragment of DNA, it has been used in pathogen diagnostics. With the increasing amount of sequencing data available, it is literally possible to design qPCR assays for every microorganism (groups and subgroups of microorganisms, etc.) of interest. The main advantages of qPCR are that it provides fast and high-throughput detection and quantification of target DNA sequences in different matrices.
A range of targets, including species of plants or animals used as food ingredients, foodborne bacteria or viruses, genetically modified organisms and allergens, even in highly processed foods, can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses.
Advantages of RT- PCR
- The conventional PCR method and other conventional techniques are expensive compared to real-time PCR
- Real-time PCR gives results in express time with the average duration ranging from 30 minutes to 2 hours
- The quantitative real-time PCR method is not only sensitive but specific and efficient as well.
- The quantitative element of PCR confirms the analytes through the melting curve analysis. Analysts can measure and quantify the number of amplicons generated and how many non-specific or primer-dimers formed during the PCR reaction by doing the melting curve analysis. The technique can quantify the template DNA or RNA present in the sample.
- There is no requirement for post PCR, but data processing is required in the quantitative real-time PCR.
Testing Method of Food Adeltaration by qPCR
Conventional methods for the detection of pathogens and other microorganisms are based on culture methods, but these are time consuming and laborious, and are no longer compatible with the needs of quality control and diagnostic laboratories to provide rapid results (Perry et al., 2007). In contrast, qPCR is a specific and sensitive alternative that can provide accurate results, and this thus opens a lot of possibilities for the direct detection of microorganisms in a food product. The targets in the foods are DNA or RNA of pathogens, as spoilage microorganisms; DNA of moulds that can produce mycotoxins; DNA of bacteria that can produce toxins; and DNA associated with trace components (e.g. allergens, like nuts) or unwanted components for food authenticity (e.g. cows’ milk in goats’ milk cheese). However, when PCR is applied for detection of pathogens in food products, some problems can be encountered, although many of these can be solved by the use of suitable sample preparation methods (Lantz et al., 1994; Hill, 1996).
Sample preparation is an important factor for PCR analysis and PCR sensitivity, especially in the direct implementation of PCR to complex foods. Sample treatment prior to PCR is also a complex issue. This mainly arises because of the need to concentrate the target DNA or RNA into the very small volumes used, which are usually 1 μl to 10 μl for PCR samples, and the presence of any PCR inhibitory substances in the samples
DNA or RNA extraction is the first step in the analysis process, and the sample quality is probably the most important component to ensure the eproducibility of the analysis and to preserve the biological meaning. Nowadays, it is relatively easy to isolate DNA at very high qualitative and quantitative
yields. Most procedures use commercial extraction kits, and depending on the food matrix, these can provide satisfactory results as supplied, or after some modifications. Different commercial kits are also available for biochemical DNA extraction.
PCR Detection of Food-borne Bacteria
There are numerous PCR-based methods for the detection of microorganisms cited in the scientific literature. There are also a number of commercially available PCR-based assays that have the convenience of providing most of the reagents and controls that are needed to perform the assay, and which appear to have high sensitivity for detecting microorganism contamination.
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