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

Xiaohan Yang; Xue Zhang; Yu Wang; Hui Shen; Ge Jiang; Jingquan Dong; Panpan Zhao; Song Gao

doi:10.3389/fmicb.2020.586981

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

Front Microbiol. 2020 Nov 6:11:586981. doi: 10.3389/fmicb.2020.586981. eCollection 2020.

Authors

Xiaohan Yang¹, Xue Zhang¹, Yu Wang¹, Hui Shen², Ge Jiang², Jingquan Dong¹, Panpan Zhao³, Song Gao¹

Affiliations

¹ Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, China.
² Jiangsu Institute of Oceanology and Marine Fisheries, Nantong, China.
³ Key Laboratory of Zoonosis Research by Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.

Abstract

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.

Keywords: Vibrio vulnificus; extracellular metalloprotease; rapid detection; real-time recombinase polymerase amplification; recombinase polymerase amplification.