Affiliation:
1. Ningbo Institute of Life and Health Industry University of Chinese Academy of Sciences Ningbo China
2. Hwa Mei Hospital University of Chinese Academy of Sciences Ningbo China
3. Key Laboratory of Diagnosis and Treatment of Digestive System Tumors of Zhejiang Province Ningbo China
Abstract
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.
Funder
Ningbo Clinical Research Center for Digestive System Tumors
Zhejiang Provincial Public Welfare Technology Program of China
Publisher
Oxford University Press (OUP)
Subject
Applied Microbiology and Biotechnology,General Medicine,Biotechnology
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