立氏立克次体实时荧光定量聚合酶链反应检测方法的建立

Study on the development of a real-time quantitative polymerase chain reaction assay to detect Rickettsia rickettsii

目的采用TaqMan-MGB探针建立立氏立克次体实时荧光定量聚合酶链反应(PCR)检测方法。方法依据立氏立克次体外膜蛋白B基因序列设计引物和探针,以克隆的ompB基因片段为DNA模板,在ABI 7900型荧光定量PCR检测仪上建立实时荧光定量PCR检测方法。结果建立的定量标准曲线Ct值与模板拷贝数呈线性关系(R2=0．996),最低检测浓度为5拷贝／μl;用荧光定量PCR方法检测其他相关立克次体和常见非立克次体病原菌,检出结果均为阴性。用该方法检测立氏立克次体感染的豚鼠血液标本、小鼠脾脏标本及细胞培养标本,检测的结果与立氏立克次体感染相关。结论研究建立的检测立氏立克次体实时荧光定量PCR方法具有高特异性和高敏感性,适用于快速检测各种样本中的立氏立克次体和立氏立克次体感染早期的实验室诊断。

Objective To develop a real-time quantitative polymerase chain reaction（PCR） assay for detecting Rickettsia rickettsii. Methods The primers and TaqMan MGB probe were designed according to the ompB gene of R, rickettsii. A DNA fragment of ompB gene amplified from R. rickettsii by PCR was used as a standard template for the development of the method. Results 5 copies of ornpB fragments of R. rickettsii were detected. The genomic DNA of R. rickettsii was detected by the developed quantitative PCR assay. However, the genomic DNA from another rickettsial or bacterial agent was not determined. Through this developed method, the positive results were obtained from the animals and cells, artificially infected with R. rickettsii. Conclusion The real-time quantitative PCR assay seemed to be highly sensitive and specific which might be used to rapidly detect R. rickettsia DNA in various samples and to early diagnose patients infected by R. rickettsii.