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

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.

Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips

“…The principle of the duplex detection biosensor for V. cholerae and V. vulnificus was based on duplex RPA amplification of the target gene fragments followed by visualization of the amplification products on a three-segment LFS ( Figure 1 ). The lolB gene of V. cholerae and empV gene of V. vulnificus had been used as molecular markers for detection of these two bacteria in previously reported assays [ 17 , 18 ]. In this study, these target gene fragments were specifically amplified in RPA reactions with chemically labeled primers and probes ( Figure 1 a).…”

“…In this study, they were selected as the labeling groups of the probes to facilitate visualization of the RPA amplification products on the strip through antibody-antigen interactions ( Figure 1 c). We designed primers and probes according to previously established RPA detection assays [ 17 , 18 ] ( Table 2 ), and selected FITC labeling for V. vulnificus (probe VV-P1) and DIG labeling for V. cholerae (probe VC-P1) to start with.…”

“…The primer and probe sequences used in this study were derived from previously reported RPA-based individual assays for V. cholerae and V. vulnificus detections ( Table 2 ) [ 17 , 18 ]. The target genes for V. cholerae and V. vulnificus were the outer membrane lipoprotein gene lolB (GenBank accession number AP014524.1) and the metalloprotease gene empV (GenBank accession number U50548.1), respectively.…”

“…and empV gene of V. vulnificus had been used as molecular markers for detection of thes two bacteria in previously reported assays [17,18]. In this study, these target gene frag ments were specifically amplified in RPA reactions with chemically labeled primers an probes (Figure 1a).…”

“…RPA reactions can amplify DNA targets isothermally under relatively low temperatures and are more convenient for on-site detections than LAMP [ 15 , 16 ]. For more on-site detection choices, RPA assays targeting lolB (outer membrane lipoprotein) gene of V. cholerae and empV (extracellular metalloproteinase) gene of V. vulnificus have been developed [ 17 , 18 ]. These molecular detection methods have improved the rapidity, sensitivity, and accuracy of the detection practice of these two important Vibrio pathogens, and have made good contributions to food safety and public health.…”