摘要
目的采用TaqMan-MGB探针建立检测莫氏立克次体的实时荧光定量PCR。方法依据莫氏立克次体外膜蛋白B基因(ompB)设计引物和探针,以克隆的ompB作模板建立实时荧光定量PCR方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.995);该方法能检出<10拷贝的莫氏立克次体DNA,敏感性为普通PCR的1000倍。用该方法检测莫氏立克次体DNA,结果为阳性,但是检测其他相关细菌DNA的结果均为阴性。用该方法检测莫氏立克次体感染豚鼠血标本,50%样本检测为阳性,而普通PCR检测结果均为阴性。结论该实时荧光定量PCR具有很高的特异性和敏感性,适合于快速检测样本中微量莫氏立克次体DNA,可用于临床实验室快速确诊地方性斑疹伤寒。
Objective To develop a real-time quantitative polyrnerase chain reaction （PCR） for detection of Rickettsii mooseri. Methods The primers and TaqMan-MGB probes were designed based on the gene encoding outer membrane B （ompB） of R. mooseri, and the real time quantitative PCR was developed with them as well as DNA template of cloned ompB. Ten-fold dilutions of ompB were determined by the real-time quantitative PCR assay to evaluate its sensitivity and precision; the genomic DNAs of R. mooseri and other bacterial agents were detected by the assay to confirm its specificity. Results The standard curve in the real-time quantitative PCR assay was drawn with ompB, and the relationship between the values of threshold cycle （Ct） and the DNA copy number found to be was linear （r=0. 995）. The minimal number of ompB that could be detected by the real-time PCR was less than 10 copies and its sensitivity was approximately one thousand times higher than that of the conventional PCR for the detection of R. mooseri. The genomic DNA of R. mooseri was found to give positive result, and that of all the other bacterial agents were found to give negative results in the real-time PCR assay. Approximate 50% of blood samples from guinea pigs infected with R. mooseri were found to be positive in the real-time assay, while on the contrary the same samples were found to be negative with the conventional PCR assay. Conclusion It is suggested that the real-time PCR is highly specific and sensitive for determination of R. mooseri. It may be adapted to detect traces of R. mooseri DNA in blood samples of patients suspected to be suffering from endemic typhus.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第10期1054-1056,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家"十五"科技攻关项目(2003BA712A04-07)
