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- Title
Development and evaluation of real‐time recombinase polymerase amplification assay for rapid and sensitive detection of Vibro mimicus in human plasma samples.
- Authors
Zhu, Peng; Huang, Zuoan; Xiong, Zi; Guo, Shiyu; Zhang, Shun; Cai, Ting
- Abstract
Aim: We aimed at developing a fast and accurate method to detect Vibrio mimicus using real‐time recombinase polymerase amplification assay. Methods and Results: Specific primers and probe were designed to target V. mimicus haemolysin (vmh) gene. Target DNA was successfully amplified at 41°C within 20 min. The method exhibited a high level of specificity and the sensitivity was 2.1 × 102 copies/25 μl or 8.4 copies/μl, which is in line with real‐time polymerase chain reaction (PCR). The calibration curve plotted by the second‐order polynomial regression showed better than the linear curve, as the correlation coefficient was raised to 0.9907, which suggested that the second‐order polynomial regressions might be considered to apply to the quantification of real‐time recombinase polymerase amplification (RPA). The limit of detection (LOD) was predicted to be 77 copies/25 μl or 3 copies/μl by a probit model. The limit of quantification (LOQ) was calculated to be 28 copies /25 μl or 1 copies/μl by a receiver operating characteristic (ROC) curve, which firstly make LOQ could be available to real‐time RPA. For the performance of the real‐time RPA in plasma samples, the detection sensitivity of real‐time RPA was as good as the real‐time PCR. For pretreatment of plasma samples, the boiling method was better than using kits, as it further shortened the time of the real‐time RPA in detecting V. mimicus. Conclusions: The real‐time RPA assay developed in our study shows multiple advantages over currently available DNA diagnostic method, including a quicker time‐to‐result for a single sample, requiring minimal infrastructure and technical support and being tolerant to inhibitors in plasma samples. Significance and Impact of the Study: The real‐time RPA assay developed here is a potentially valuable tool for point‐of‐care (POC) diagnosis of V. mimicus infection in endemic field, especially in the resources‐limited settings, as combined with portable devices.
- Subjects
RECOMBINASES; RECEIVER operating characteristic curves; POLYMERASE chain reaction; ENDEMIC diseases; DENATURING gradient gel electrophoresis; DETECTION limit
- Publication
Journal of Applied Microbiology, 2022, Vol 133, Issue 3, p1650
- ISSN
1364-5072
- Publication type
Article
- DOI
10.1111/jam.15666