Development of recombinase polymerase amplification combined with lateral flow dipstick assay to detect hemolysin gene of Vibrio vulnificus in oysters

Authors: PARK, SEONG - Coastal Research And Extension Center; CHANG, SAM - Coastal Research And Extension Center

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2022
Publication Date: 4/15/2022
Citation: Park, S., Chang, S. 2022. Development of recombinase polymerase amplification combined with lateral flow dipstick assay to detect hemolysin gene of Vibrio vulnificus in oysters. Journal of Food Protection. 85(12):1716–1725. https://doi.org/10.4315/JFP-21-455.
DOI: https://doi.org/10.4315/JFP-21-455

Interpretive Summary: Vibrio vulnificus is a type of bacteria found in estuarine waters all over the world. If people eat raw or undercooked oysters that are contaminated with this bacteria, it can lead to severe infections. Right now, there are methods to detect this bacteria, but they require specialized equipment and trained operators. In this study, researchers have come up with a new and simple way to detect the presence of Vibrio vulnificus quickly and accurately, even without the need for specialized equipment. They used a specific gene called "vvh" to identify the bacteria. The method they developed, called "recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD)," could detect tiny amounts of the bacteria in just 30 minutes. It worked well at a wide range of temperatures. The test they created was very specific to Vibrio vulnificus and did not react to other similar bacteria that could be found in food. They compared their new method with other common tests and found that it performed just as well as the most accurate one. In conclusion, this new RPA-LFD method could be a valuable tool to quickly detect Vibrio vulnificus in oysters, especially in places where resources are limited or in field conditions. By identifying the presence of this harmful bacteria early on, it could help prevent infections and ensure the safety of seafood consumption.

Technical Abstract: Vibrio vulnificus inhabits estuarine waters around the world and can cause severe infections in people who eat contaminated raw or undercooked oysters. Although current detection methods are sensitive and specific, there are continuous demands for the development of rapid and accurate methods without a trained operator and equipment in the field conditions. Herein, we developed a simple and rapid method by detecting the hemolysin (vvh) gene of V. vulnificus by using recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD). The RPA-LFD could detect 100 fg of DNA (P , 0.05) and 20 CFU of V. vulnificus per reaction within 30 min (P , 0.01) and showed the result with incubation temperature ranges from 30 to 458C (P , 0.001). The test was specific only to V. vulnificus and was not responsive to 10 other closely related Vibrio species and 18 foodborne pathogenic bacteria. Compared with PCR, quantitative PCR, and colony hybridization assays by using naturally contaminated oyster samples, our RPA-LFD showed the same detection ability as quantitative PCR assay. Therefore, the current RPA-LFD would be a valuable tool to detect V. vulnificus in oysters, especially in field conditions.