实时荧光定量PCR检测斑点热立克次体方法的建立

Establishment of a TaqMan-based real-time PCR method of spotted fever group rickettsiae

目的建立斑点热群立克次体(SFGR)TaqMan实时荧光定量PCR(qPCR)快速检测方法。方法根据日本斑点热立克次体外膜蛋白A(ompA)基因序列设计特异性引物和探针,建立基于TaqMan探针的实时荧光定量PCR检测方法,对其特异性、灵敏性、重复性进行检测,用建立的此检测方法和常规的巢式PCR方法同时对临床收集的80份患者血标本进行检测并比较两者结果。结果成功建立了检测SFGR的实时荧光定量PCR方法,标准曲线的循环阈值与模板拷贝数均呈良好的线性关系(r>0.99),且在检测SFGR核酸样本时具有良好的灵敏度、特异性和重复性。结论该研究建立了基于TaqMan探针检测SFGR的实时荧光定量PCR检测方法,为临床实验室快速确诊SFGR疾病提供了快速、有效的技术手段。

Objective To establish a TaqMan real-time fluorescent quantitative PCR of spotted fever group rickettsiae(SFGR). Methods According to the ompA gene sequence of Rickettsia.japonica,the specific primers and probes were designed to develop the TaqMan-based Real-time PCR method. The specificity, sensitivity and repeatability of the established method were verified. The established TaqMan real-time PCR method was used to detect SPGR infection in 80 clinic blood samples collected in Anhui Province and compared with the nested PCR. Results The real-time quantitative PCR method for detecting SPGR was successfully established. The cyclic threshold of the standard curve and the template copy number showed a good linear relationship(r>0.99). This method had strong specificity, high sensitivity and good repeatability in the detection of the spotted heat rickettsial nucleic acid samples. Conclusion This study establishes a real-time quantitative PCR assay based on TaqMan probe to detect SPGR, which provides a rapid and effective technical means for rapid diagnosis in clinical laboratories.