Yang Huan-Lan, Wei Shuang, Gooneratne Ravi, Mutukumira Anthony N, Ma Xue-Jun, Tang Shu-Ze, Wu Xi-Yang
a Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China.
b Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510632, China.
Can J Microbiol. 2018 Apr;64(4):223-230. doi: 10.1139/cjm-2017-0504. Epub 2018 Jan 19.
A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 10 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.
开发了一种使用重组酶聚合酶和内部扩增对照（IAC）的新型RPA-IAC检测方法，用于副溶血性弧菌的检测。基于副溶血性弧菌toxR基因的编码序列设计了特异性引物。重组酶聚合酶扩增（RPA）反应在37°C的恒定低温下进行20分钟。通过使用63株弧菌菌株和10种非弧菌细菌验证了检测的特异性。此外，采用竞争性IAC来避免假阴性结果，其与靶序列同时进行共扩增。该检测方法的灵敏度确定为3×10 CFU/mL，明显比既定的PCR方法更灵敏。然后使用该方法检测从当地市场采集的海鲜样品。53种不同的生鲜海鲜中有7种被检测为副溶血性弧菌阳性，这与使用传统培养方法和生化检测获得的结果一致。这种新型RPA-IAC检测方法为副溶血性弧菌提供了一种快速、特异、灵敏且更便捷的检测方法。
