A Real-Time Recombinase Polymerase Amplification Method for Rapid Detection of Vibrio vulnificus in Seafood

• Main Authors: Xiaohan Yang, Xue Zhang, Yu Wang, Hui Shen, Ge Jiang, Jingquan Dong, Panpan Zhao, Song Gao
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2020-11-01
• Series: Frontiers in Microbiology
• Subjects: Vibrio vulnificus; real-time recombinase polymerase amplification; rapid detection; recombinase polymerase amplification; extracellular metalloprotease
• Online Access: https://www.frontiersin.org/articles/10.3389/fmicb.2020.586981/full

As an important foodborne pathogen, Vibrio vulnificus gives a significant threat to food safety and public health. Rapid and accurate detection methods for V. vulnificus are required to control its spread. The conventional detection methods are time-consuming and labor-intensive, while the polymerase chain reaction (PCR)- and quantitative PCR (qPCR)-based methods are limited because of their dependence on laboratory equipment. Nucleic acid isothermal amplification technologies have been applied to develop simpler assays. In this study, a rapid detection method based on real-time recombinase polymerase amplification (RPA) targeting the extracellular metalloprotease (empV) gene of V. vulnificus has been established. The method finished the detection in 2–14 min at 39°C with good specificity. The limit of detection was 17 gene copies or 1 colony-forming unit (CFU) per reaction, or 1 CFU/10 g of spiked food with enrichment. In a clinical sample detection test, the results of real-time RPA were 100% consistent with bioassay and qPCR. Moreover, the method could resist the effect of food matrix and could tolerate crude templates. The real-time RPA method established in this study is rapid and simple and has the potential to be widely applied for V. vulnificus detection in food safety control.