Dataset Information

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.

SUBMITTER: Yang X

PROVIDER: S-EPMC7677453 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Yang Xiaohan X Zhang Xue X Wang Yu Y Shen Hui H Jiang Ge G Dong Jingquan J Zhao Panpan P Gao Song S

Frontiers in microbiology 20201106

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 ...[more]

PMID: 33240242

Similar Datasets

Project description:Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R2 value was 0.983 for the concentration range 0.2 ng (4.1 × 107 DNA copies) to 2.0 fg (4.1 × 101 DNA copies). For the pUC57-ema gene DNA, the R2 value was 0.993 for the concentration range 0.2 ng (5.26 × 107 DNA copies) to 2.0 fg (5.26 × 102 DNA copies). For the pUC57-Bc18S gene DNA the R2 value was 0.976 for the concentration range 2.0 ng (4.21 × 108 DNA copies) to 2.0 fg (4.21 × 102 DNA copies). For the pUC57-Te18S gene DNA, the R2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 108 DNA copies) to 2.0 fg (4.16 × 102 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.

Dataset Information

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

Publications

A Real-Time Recombinase Polymerase Amplification Method for Rapid Detection of <i>Vibrio vulnificus</i> in Seafood.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets