应用Taqman实时PCR法检测禽肉中沙门菌的研究

Study on detection of Salmonella in poultry samples by real-time PCR with Taqman probes

目的 建立敏感快速的检测禽肉中沙门菌的实时PCR法.方法 以invA基因为基础,建立RT-PCR法并验证其特异性.选用肠炎沙门菌CMCC 50041,制备不同浓度的纯菌液,用RT-PCR进行检测,制作标准曲线并计算扩增效率.进行人工染菌实验,染菌量分别为每25 g鸡肉样本1、10、102、103、104、105和106 CFU.分别在增菌0、4、8、12、18 h取1 ml培养液,提取DNA进行RT-PCR检测,并同时用PCR和传统方法进行检测,比较3种方法检测的敏感性和特异性.采集16份市售整鸡样本,用这3种方法进行检测,进一步比较三者的阳性检出情况.结果 建立的RT-PCR法特异性好,对纯菌液的最低检测限为5.2×103 CFU/ml,在5.2×103～ 5.2×1010 CFU/ml范围内,菌浓度的对数值和检测结果的Ct值线性关系良好,R2=0.999.人工染菌样本在增菌12 h后,RT-PCR法检出限可达l CFU/25 g,与PCR检测的敏感性一致,比传统方法的检出限敏感10倍.根据增菌12 h的检测结果,建立了RT-PCR样本标准曲线.采用RT-PCR检测16份禽肉样本,检出7份阳性,与PCR一致,高于传统方法（5份阳性）.根据样本标准曲线,对检测样本进行定量分析,确定了阳性样本中初始含量菌.结论 所建立的RT-PCR具有快速简便、敏感特异等优点,适用于禽肉中沙门菌的快速定量检测.

Objective To develop a RT-PCR method for a rapid and sensitive detection of Salmonella in poultry samples.Methods The RT-PCR method was established and its specificity was testified on the basis of invA gene.Serial 10-fold diluted pure suspension culture of CMCC 50041 was detected by RT-PCR,the standard curve was constructed and the amplification efficiency was calculated.Artificially contaminated experiment was done,six artificially-inoculated samples containing final concentration of Salmonella CMCC 50041 （1,10,102,103,104,105 and 106 CFU per 25 g poultry samples） were prepared respectively.All the samples were incubated for 0,4,8,12 and 18 h and the DNA was extracted for RT-PCR detection,meanwhile by PCR detection and the traditional method.The sensitiveness and specificity were compared among the three methods.At the same time,16 samples of retail whole poultry were collected from markets and detected by the above three methods as well,and thereby to further compare the positive detection among the three methods.Results The established RT-PCR method was specific for the detection of Salmonella.The sensitivity was 5.2 × 103 CFU/ml for pure Salmonella culture without enrichment.Correlation coefficients of standard curves constructed using the Ct versus log value of concentration of Salmonella showed good linearity over a 8-log dynamic range （5.2 × 103-5.2 × 1010 CFU/ml）,with the R2 at 0.999.RT-PCR detection limit for artificially contaminated samples after enriching for 12 h was 1 CFU/25 g sample,which was the same with the limit of PCR and 10 times more sensitive than the limit of traditional method.Standard curve of sample after enrichment for 12 h was established.Seven of 16 samples were detected positive by RT-PCR,which were also tested positive by PCR,while only five samples were positive by traditional method.The positive ones were quantitatively analyzed using standard curve of sample and determined the initial Salmonella numbers of CFU/25 g.Conclusion The established RT-PCR technology was simple,rapid,sensitive and specific,which was suitable to quickly detect Salmonella in poultry samples.