How to Quickly Detect Vibrio spp. in Seafood Using PCR Technology

Standard methods are also labor intensive and rely on microbiological/biochemical identification. For seafood processors and inspections, current methods require at least three to five days for results and subjective interpretation for the screening of negative samples.

Food testing laboratories in the seafood industry are in need of a fast and accurate method to reliably detect the three main Vibrio species. An easy-to-use and rapid method that can reliably report results would allow seafood to safely get to market faster.

Real-Time PCR Detection

The advantages of real-time PCR are highlighted when used for the detection of Vibrio because of the challenges outlined above, such as the background flora naturally present in seafood matrices and the enrichment protocol challenges.

Since its invention, real-time PCR technology has been greatly improved so that it is more stable, accurate, and rapid for specific applications. As the technology evolved, new chemistries were developed based on fluorescence detection.

This evolution allowed for real-time PCR kits to achieve a high level of specificity and sensitivity when detecting Vibrio. Each test well can be used to detect all three important strains of the pathogen at the same time, decreasing the time it takes to get a result. A PCR kit may be able to detect the pathogen in 94 samples in less than two hours, rather than the typical three to five days. Further, the workflow is often optimized to be simple and user friendly.

While real-time PCR methods often offer quicker turnaround times than many of the standard methods, they can be prone to false positives due to free DNA from dead cells found in the sample. Emerging PCR-based methods should address this limitation.

Recently, Bio-Rad Laboratories received AOAC validation for its iQ-Check Vibrio assay. The assay uses a single-step enrichment followed by real-time PCR for the multiplex detection of VC, VP, and VV. This method provides rapid qualitative detection and differentiates among all three strains in seafood products. The solution also has an optional Free DNA Removal Solution that can address ambiguity caused by dead cell DNA by removing free DNA in the sample with a simple non-toxic protocol, while the intact DNA in living cells remains unaffected.

This method was evaluated and approved by the AOAC Performance Tested Methods (PTM 032002) program. Results of the AOAC-PTM validation study demonstrated no differences between the iQ-Check Vibrio method and the U.S. FDA BAM Vibrio reference method. The assay and the Free DNA Removal Solution were validated for use with 125-gram test portions of cooked and raw shrimp, raw mussels, raw oysters, and raw tuna. The assay was approved for use with Bio-Rad Vibrio Enrichment Broth (after a seven-to-nine hour enrichment period) and alkaline phosphate water (after a six- to 18-hour enrichment period), giving the user flexibility to optimize the method to their lab workflow, while significantly cutting down the traditional three to five days it takes to get results with standard methods.

Rapid methods like this one can greatly aid in outbreak investigation and management of public health concerns. The ability to obtain results in a shorter amount of time, particularly when it comes to pathogens such as Vibrio species, can be critical in reducing the impact from a food safety event.

Pastori is an international product manager at Bio-Rad Laboratories. Reach him at [email protected]. Wang is a field marketing specialist at Bio-Rad Laboratories. Reach him at [email protected].