鼠疫耶尔森菌TaqMan探针实时荧光定量PCR快速检测

目的 建立一种快速检测鼠疫耶尔森菌的方法.方法 以编码F1抗原的基因caf1为靶序列,人工合成基因序列,制备重组质粒作为鼠疫耶尔森菌检测的标准样品；用实时荧光定量聚合酶链反应(fluorescent quantitative polymerase chain reaction,FQ-PCR)技术,针对pMT1质粒上的caf1基因设计引物和TaqMan荧光探针,进行实时FQ-PCR检验,评价该方法的准确性、敏感性及特异性.建立鼠疫耶尔森菌TaqMan探针实时FQ-PCR检测方法.结果 本研究成功构建了质粒pUC57-caf1,引物、探针特异性良好,标准曲线在5.03×102～5.03×107拷贝数之间,具有较好的线性关系,相关系数1.0.实时FQ-PCR检验最低可检测出5.03×102拷贝数的重组质粒,灵敏度比普通PCR高100倍.特异性检测试验可选择性检测出鼠疫耶尔森菌,与其他细菌无交叉反应,结果与普通PCR一致.对同一浓度的重组质粒进行15个平行样品的重复性试验,Ct值标准差为0.28.结论 建立TaqMan探针实时FQ-PCR检测技术,能够快速、准确、特异的检测鼠疫耶尔森菌,为鼠疫监控、诊断提供技术支持.

Rapid detection of Yersinia pestis with TaqMan probe by real time fluorescent quantitative

Objective To establish a rapid detection method of Yersinia pestis. Methods The recombinant plasmid of Yersinia pestis was synthesized with target sequence carl of F1 antigen synthetic and used as the standard sample. Primers and the TaqMan fluorescent probe were designed for carl of pMT1 plasmid. Real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） technology was performed to evaluate accuracy, sensitivity and specificity. Finally, real time FQ-PCR method with Yersinia pestis TaqMan probe was built. Results The pUC57-cafl plasmid was successfully constructed with a good linear relevant standard curve between the 5.03×10^2and 5.03×10^7 copies and perfect primers, and probe specificity. The correlation coefficient was 1.0. The method could detect a minimum recombinant plasmids amount a- bout 5.03 ~ 102 copies which was as a hundred folds sensitive as ordinary PCR coincident with ordinary PCR results, FQ- PCR specificity test could detect Yersinia pestis selectively with no cross reaction together with other germs. Reproducibility tests for 15 parallel samples under the same conditions obtained a 0.28 Ct standard deviation. Conclusions The TaqMan probe real-time fluorescent quantitative PCR detection technology, detecting Yersinia pestis quickly, accurately and specifically, has been built. It gives a technical support for plague monitoring and diagnosis.