Real-Time Recombinase Polymerase Amplification Assay for the Detection of Vibrio cholerae in Seafood

Tang, Yuyi; Cao, Yunqing; Yu, Yongxin; Yan, Shiqiang; Wang, Yongjie; Pan, Yingjie; Zhang, Weijia

doi:10.1007/s12161-017-0820-7

Cited by 4 publications

(9 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

Section: Principle Of the Biosensormentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Wang

Liao²,

Ma³

et al. 2021

Biosensors

View full text Add to dashboard Cite

Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 101 copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 °C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%