Real-Time PCR探针法定量检测沙门菌方法的建立及应用

目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。

Establishment and application of real-time PCR for quantitative detection of Salmonella spp.

To establish real-time PCR for quantitative detection of Salmonella spp.,a pair of primers and fluorescent probe were designed according to stn gene sequence of Salmonella spp.,and then a real-time PCR method was successfully established to detect Salmonella spp.through the exploration of reaction system and reaction conditions.Results demonstrated that the method is specific and sensitive.Salmonella spp.detected by this real-time PCR was positive while non-Salmonella one was negative.The correlation coefficient of standard curve（R2）was 0.993 and the detecting threshold was up to 5 CFU.After the detection of 108 chicken feces,50 chicken meats and 58 water samples were detected with the this method and the positive rates were 3.7%（6/108）,4%（2/50）and 3.4%（2/58）respectively.The result was consistent with results from traditional microbiology method.The method is simple,rapid,specific and sensitive,and this study made a good foundation for rapid detection of Salmonella spp.in the environment study or disease diagnosis.