qPCR Assays for Microbial Detection and Food Safety

Real-time PCR, qPCR, or qRT-PCR analysis is a well-established approach for detecting and quantifying different microbial agents in food safety, veterinary, and clinical diagnostics. PCR is a relatively simple technique. Some variations of this method include copy number qPCR and ddPCR assay. However, qPCR has specific challenges that developers and users should address before starting assessments. These concerns incorporate understanding the PCR principle, use of correct definitions and terminology, difficulties with data presentation and interpretation, and comprehending the performance limitations in microbial diagnostics and safety testing. This article focuses on the different aspects of qPCR assays used in microbial detection and food safety assessments.

qPCR testing for microbial and food safety assessments

Researchers employ two primary strategies to visualize DNA amplification in real time: fluorescently labeled oligonucleotide probes and non-specific fluorescent DNA dyes. Both these strategies were developed in parallel and are employed for detecting pathogens. However, probe-based approaches prevail, due to higher specificity through additional oligonucleotides and lower susceptibility to visualize non-specific products.

As PCR can amplify a specific DNA fragment, it can diagnose pathogens. With an increasing amount of data available on DNA sequencing, researchers can literally develop qPCR assays for all microorganisms of interest. The primary advantage of qPCR assays is that they offer high throughput and rapid detection and quantification of target sequences in diverse study matrices. Simultaneous visualization and amplification of new DNA amplicons facilitates a lower amplification period. qPCR assays reduce cross-contamination as no additional sample manipulation is needed after amplification. Other advantages include broader dynamic range and multiplexing capacities of different targets in a single reaction volume. Most importantly, multiplexing ability is critical for detecting and quantifying analytes that rely on incorporating internal amplification controls.

qPCR assays not only detect but also determine the presence of alleles and genes, for example, antimicrobial resistance profiling, typing of isolates and strains, and toxin production. However, the presence of resistant genes or toxin production does not necessarily mean their production or expression. Hence, biochemical and phenotypic assessments are critical for correlating qPCR-based typing results. Microbial diagnostics need unique considerations for detecting and quantifying parasitic, bacterial, and viral agents. These considerations largely depend on the target, interpreting results, cultivability, and clinical significance of data.

qPCR assays are crucial for detecting, quantifying, and typing viral pathogens. The primary advantage of qPCR testing is that culture methods for veterinary and clinical viral detection are lengthy. On the other hand, approaches such as ELISA assays offer low specificity and sensitivity and are not universally available. qPCR assays offer appropriate specificity and sensitivity. qRT-PCR analysis of viral load indicates a response against antiviral therapies. Due to these reasons, qPCR has now become a vital tool in viral diagnostics.

In food safety testing, all international standards rely on determining pathogens using conventional culture methods. qPCR testing offers an excellent alternative to standard culture approaches, as it provides reliable quantification and detection for multiple pathogens and offers several other advantages discussed above. However, qPCR assays have limitations in terms of assay sensitivity. As culture media approaches are based on bacterial multiplication during the pre-enrichment stage, DNA isolation samples generally have low target bacterial count. These limitations result in one of the most significant disadvantages of qPCR assays, which is the inability to distinguish between dead and live cells.

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The usage of qPCR assays is hence limited to the identification of antimicrobial resistance, typing bacterial strains, and detection and quantification of strains in raw and non-processed food. Notably, processed food may still contain DNA that can be amplified even after all pathogenic bacteria are devitalized and the food is safe for consumption. Companies overcome this issue by adding a sample pre-enrichment step in culture media before qPCR analysis. This step may contain specific selective media for bacteria or non-selective enrichment in peptone water. This procedure is to facilitate recovery and bacterial multiplication for subsequent qPCR detection. This approach enables the dilution and elimination of PCR inhibitors in the sample, such as conservation substances and the presence of salts. Compared to food samples, DNA extraction from culture media is easier, as these samples are more heterogeneous.

Conclusion

qPCR assays are a powerful technology in food safety and microbial detection. qPCR services have significant potential in the detection, typing, and quantification of parasites and viruses. Today, qPCR and PCR services are replacing culture techniques in bacterial diagnostics, particularly when sensitive and rapid testing is needed.

Laboratories perform qPCR standardization according to different rules. These discrepancies are largely due to the lack of standard protocols defined for basic qPCR functional parameters. There are variations in standardization and validation approaches of qPCR assays across food safety, veterinary testing, and clinical sciences. Hence, a collaborative effort is vital to unify the validation and standardization protocols for improving the quality of qPCR Bioanalytical Services in microbial detection and food safety testing.