猪源产肠毒素性大肠杆菌耐热肠毒素STⅡ基因荧光定量PCR检测试剂盒的研制

A real-time quantitative PCR kit for detection of enterotoxigenic Escherichia coli heat-stable toxin STⅡ gene from porcine

为建立猪源产肠毒素性大肠杆菌（ETEC）的快速准确诊断方法,根据Gen Bank收录的ETEC耐热肠毒素（STⅡ）基因序列设计1对特异性引物,采用SYBR GreenⅠ荧光染料建立ETEC荧光定量PCR诊断试剂盒,并验证该检测法的敏感度、特异性、重复性及试剂盒长期保存的稳定性。结果显示：该诊断试剂盒的最低灵敏限度达1.0×10^1拷贝/μL,特异性强,对羊源、牛源产肠毒素性大肠杆菌基因未检出;用该试剂盒检测猪场分离的149株临床样品,同时与传统的分离培养后普通PCR比较,该方法检出率高;试剂盒于-20℃保存3-6个月对阳性标准品拷贝数无影响。本研究研制的试剂盒在临床检测大肠杆菌比传统检测方法快且结果表明建立的试剂盒适合于ETEC的检测,为该病的快速诊断和防治提供基础。

In order to establish a rapid and accurate method for diagnosing porcine enterotoxigenic Escherichia coli（ ETEC）,a pair of specific primers were designed here according to the gene sequence of heat-stable enterotoxin of ETEC and SYBR Green I was used to build a diagnostic kit for ETEC. The research also verified the sensibility,specificity,repeatability and stability of the detection method for long term maintenance. The results showed that the lowest limit of sensibility of this diagnostic kit was 1. 0× 10^1copies/ul and high specificity,but it could not detect other genes from lamb and calf in ETEC. This diagnostic kit showed a high detection rate by detecting 149 clinical samples,compared with ordinary PCR. And preservation of the kit at-20℃ for 3 to 6 months had little effect on the copies of the positive standard.The diagnostic kit constructed here was applicable to detecting ETEC in pigs,and provided basis for quick diagnosis and prevention of ETEC infection.