双重实时荧光聚合酶链式反应检测棘鳞蛇鲭和异鳞蛇鲭

Development of a duplex real-time polymerase chain reaction for Ruvettus pretiosus and Lepidocybium flavobrunneum

目的建立检测棘鳞蛇鲭(Ruvettus pretiosus, RP)和异鳞蛇鲭(Lepidocybium flavobrunneum, LF)两种油鱼的双重实时荧光聚合酶链式反应(PCR)方法。方法基于棘鳞蛇鲭和异鳞蛇鲭线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)基因分别设计特异性引物和探针,并对方法进行灵敏度、特异性和重复性分析,建立双重实时荧光PCR方法。结果模板量在8.4×10^5~8.4×10^0 copies/μl范围内,方法具有良好的线性关系,棘鳞蛇鲭和异鳞蛇鲭的线性回归方程分别为y=-3.18x+41.0(R^2=0.998 8)和y=-3.37x+44.5(R^2=0.998 6),方法组内相对标准偏差(RSD)为0.29%~0.84%。其中,检测棘鳞蛇鲭的组内RSD为0.29%~0.84%,检测异鳞蛇鲭的组内RSD为0.29%~0.62%。方法最低检测限为16.8 copies/反应,并且从随机购买的50份鳕鱼样品中鉴定出油鱼成分棘鳞蛇鲭。结论本试验建立的双重实时荧光PCR方法能够特异性地鉴定出棘鳞蛇鲭和异鳞蛇鲭,具有实际应用潜力。

Objective To establish a duplex real-time polymerase chain reaction(PCR) method for simultaneous detection of the Ruvettus pretiosus and Lepidocybium flavobrunneum. Methods The specific primers and probes were designed base on cytochrome C oxidase subunitⅠ gene of Ruvettus pretiosus and Lepidocybium flavobrunneum, respectively. The perfromance of method was assessed, including sensitivity, specitivity and repeatability. The method was applied to detect the Ruvettus pretiosus and Lepidocybium flavobrunneum from fish products. Results The result indicated that duplex real-time PCR method was specific to Ruvettus pretiosus and Lepidocybium flavobrunneum. The method showed good linear relationship between Ct value and sample copies in 8.4×10^5-8.4×10^0 copies/μl, and the linear regression equation were y=-3.18x+41.0(R^2=0.998 8) and y=-3.37x+44.5(R^2=0.998 6) for Ruvettus pretiosus and Lepidocybium flavobrunneum, respectively. The limit of quantification(LOQ) was 16.8 copies. The relative standard deviation(RSD) of this duplex real-time PCR was 0.29%-0.84%, the RSD was 0.29%-0.84% and 0.29%-0.62% for Ruvettus pretiosus and Lepidocybium flavobrunneum, respectively. The Ruvettus pretiosus were identified using this method from 50 codfish products. Conclusion This duplex real-time PCR was a sensitive, sepecific and stable method. It would be useful for dete-ction of the Ruvettus pretiosus and Lepidocybium flavobrunneum.