Development of Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay To Detect Hemolysin Gene of Vibrio vulnificus in Oysters

J Food Prot. 2022 Dec 1;85(12):1716-1725. doi: 10.4315/JFP-21-455.

Abstract

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 45°C (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.

Keywords: Vibrio vulnificus; Equipment-free detection; Hemolysin gene; Lateral flow dipstick; Oyster; Recombinase polymerase amplification.

MeSH terms

  • Animals
  • Hemolysin Proteins / genetics
  • Humans
  • Nucleic Acid Amplification Techniques / methods
  • Ostreidae* / microbiology
  • Real-Time Polymerase Chain Reaction
  • Recombinases
  • Sensitivity and Specificity
  • Vibrio vulnificus*

Substances

  • Hemolysin Proteins
  • Recombinases