斑疹伤寒群立克次体groEL基因荧光定量聚合酶链反应方法的建立及临床病例检测分析

Establishment of real-time PCR assay for typhus group rickettsia groEL genes and clinical case detection

目的基于斑疹伤寒群立克次体热休克蛋白gro EL基因,建立一种敏感、特异、快速检测斑疹伤寒群立克次体荧光定量PCR技术。方法通过GenBank分析比较斑疹伤寒群立克次体与其他立克次体成员groEL基因,选择斑疹伤寒群立克次体两个种即普氏立克次体及莫氏立克次体保守序列设计MGB探针及引物并进行方法学评估。同时用本法及普通PCR技术对临床收集的26例可疑斑疹伤寒患者急性期血液DNA进行检测分析。结果本研究建立的方法,除普氏立克次体及莫氏立克次体检出阳性外,其他立克次体成员检测阴性。该方法最小检出浓度为102copies/μl。标准曲线各浓度点Ct值批内、批间平均变异系数-CV%分别为1.41%和2.66%。26例可疑病例标本2例检测阳性,血液标本groEL基因分别定量为103和102copies/μl,并得到血清学结果证实。结论建立的斑疹伤寒群立克次体荧光定量PCR法具有快速、敏感和特异的优点,可用于斑疹伤寒患者标本的检测及疾病监测工作中。

Objective To establish an efficient and sensitive real-time PCR assay for the early detection of typhus group rickettsia based on the groEL gene. Methods MGB probe and specificity primers were designed based on the conserved sequences of groEL gene of R.provazakii and R.typhi and methodology evaluation were performed. Twenty six blood samples collected from probable patients with typhus were detected by the established assay. Results Twenty four non-typhus bacterial strains were tested. Specific amplification was only achieved with genomic DNA from R.provazakii and R.typhi. The lowest detection limit of the assay was about 100 copies of groEL genes. The average intra-and-inter coefficient of variations （CV） of the Ct value for different copies of groEL genes were 2.66% and 1.41% respectively. Two clinical blood samples from patients with typhus confirmed by seroconversion were detected as 103 copies/μl and 102 copies /μl respectively. Conclusion Real-time PCR assay was capable for detecting as few as 100 copies of groEL from genomic DNA and this assay is suitable to use in early diagnosis of R.provazakii and R.typhi infections.