One of the most critical success factors for all PCR designed for food safety is the accuracy and reliability of the results. This involves much more than just the specificity of the detection system. It is about “robustness”—that is, the ability to withstand changes under varying conditions. Join us for this third part of our series of articles on PCR, where we discuss how PCR are designed for robustness.
Precision is achieved by conducting inclusivity and exclusivity studies that detect the target of interest and exclude all other components without compromising detection sensitivity. The assay should be validated using multiple production batches of assay components to ensure reproducibility across material batches. Once the targetPCR has been established, it is further developed to meet the practical requirements of the industry.
The most challenging aspect of detecting microbial pathogens in food is the food matrix itself, where the requirement is to detect 1 viable organism per analytical unit (e.g., from 25 g to 375 g). No analytical method is currently capable of this on its own; instead, an enrichment procedure is required during which the target organism grows to a detectable level.It is absolutely essential to develop an accurate detection method that must perform consistently on a diverse range of food matrices, some of which may havespecific enrichment requirements. Subsequent procedures are then required to transfer and prepare the enriched sample for analysis that meets the limit of detection (LOD)for the overall analysis.
A procedure for lysing target cells in the enriched sample is required to release DNA that serves as a template for amplification in PCR. These procedures must be simple and easy to use to accommodate the high testing volumes in food testing laboratories. The complexity and number of potential variables in food testing require careful and rigorous testing of PCR during development and thereafter. The desired LOD in food matrices is verified through spiking studies following enrichment with known levels of target cells.
Field trials conducted in collaboration with laboratories and customers provide an opportunity to determine the “real-world” performance and usability of the overall analysis for the end consumer. It is also important to incorporate as many known failure scenarios as possible into product development to ensure that overall performance is minimally affected in the event of deviations from the recommended procedure.
These variations range from studies examining defects in manufacturing processes to common customer errors. These studies are important for determining the effects of inherent variables, such as the supply of test components, that can affect the final product. Data from development activities is used to develop and validate the final result.
A key requirement for the food testing market is that results must be easy to interpret and enable rapid, confident decision-making. This is achieved through the development of analytical algorithms designed to generate definitive output. A well-designed screening method must incorporate data interpretation into the design process. Testing methods based on molecular biology have high specificity, but many other variables must be built into every step of the procedure to ensure a robust and reliable analysis.