Department of Pharmacology, Hebei University of Chinese Medicine, No.326 South Xinshi Road, Shijiazhuang, 050091, Hebei, China.
College of Life Sciences, Hebei Normal University, No.20, Road E. 2nd Ring South, Yuhua District, Shijiazhuang, Hebei Province, 050024, People's Republic of China.
BMC Microbiol. 2019 Aug 13;19(1):186. doi: 10.1186/s12866-019-1562-z.
Vibrio parahaemolyticus (V. parahaemolyticus) is a leading cause of food poisoning and is of great importance to public health due to the frequency and seriousness of the diseases. The simple, timely and efficient detection of this pathogen is a major concern worldwide. In this study, we established a simple and rapid method based on recombinase polymerase amplification (RPA) for the determination of V. parahaemolyticus. According to the gyrB gene sequences of V. parahaemolyticus available in GenBank, specific primers and an exo probe were designed for establishing real-time recombinase polymerase amplification (real-time RPA).
The real-time RPA reaction was performed successfully at 38 °C, and results were obtained within 20 min. The method only detected V. parahaemolyticus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. The study showed that the detection limit (LOD) of real-time RPA was 1.02 × 10 copies/reaction. For artificially contaminated samples with different bacteria concentrations, V. parahaemolyticus could be detected within 5-12 min by real-time RPA in oyster sauce, codfish and sleeve-fish at concentrations as low as 4 CFU/25 g, 1 CFU/25 g and 7 CFU/25 g, respectively, after enrichment for 6 h, but were detected in a minimum of 35 min by real-time PCR (Ct values between 27 and 32).
This study describes a simple, rapid, and reliable method for the detection of V. parahaemolyticus, which could potentially be applied in the research laboratory and disease diagnosis.
副溶血性弧菌（V. parahaemolyticus）是食源性疾病的主要病原体之一，由于其发病率和严重性，对公共卫生具有重要意义。该病原体的简单、及时和高效检测是全世界关注的焦点。本研究建立了一种基于重组酶聚合酶扩增（RPA）的快速检测副溶血性弧菌的方法。根据 GenBank 中副溶血性弧菌的 gyrB 基因序列，设计了特异性引物和外切探针，用于建立实时重组酶聚合酶扩增（real-time RPA）。
实时 RPA 反应在 38°C 下成功进行，结果在 20 min 内获得。该方法仅检测副溶血性弧菌，与其他细菌无交叉反应，具有很高的特异性。研究表明，实时 RPA 的检测限（LOD）为 1.02×10 拷贝/反应。对于不同浓度细菌的人工污染样品，经 6 h 富集后，实时 RPA 可在蚝油、鳕鱼和乌贼中分别于 5-12 min 内检测到浓度低至 4 CFU/25 g、1 CFU/25 g 和 7 CFU/25 g 的副溶血性弧菌，而实时 PCR 则需要 35 min 以上（Ct 值在 27 到 32 之间）才能检测到。
本研究描述了一种简单、快速、可靠的副溶血性弧菌检测方法，该方法可能在研究实验室和疾病诊断中得到应用。
