TaqMan-MGB探针实时荧光定量PCR检测恙虫病东方体方法的建立

Establishment of Real-time PCR assays for detection of Orientia tsutsugamushi 56 kD gene with a TaqMan-MGB probe

目的建立敏感、特异、定量Real-time PCR方法,用于恙虫病东方体的检测。方法根据恙虫病东方体56 kD外膜蛋白基因序列设计引物和探针,建立实时荧光定量PCR检测方法。结果建立的TaqMan-MGB探针具有良好的特异性,建立的荧光定量PCR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=0.99),灵敏性评估发现每个20μlPCR反应管中只要有26个拷贝的目的基因即可被检测到,即最低检出浓度为2拷贝/μl,并且具有较好的重复性。结论建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于恙虫病东方体感染的快速检测。

Objective To develop a highly specific and sensitive Real-time PCR assay to detect Orientia tsutsugamushi.Methods A pair of primers and a TaqMan-MGB probe were designed according to the 56 kD outer membrane gene sequence.Results A linear relationship between the threshold cycle（Ct） of the quantitative Real-time PCR and the DNA copy number was demonstrated（r=0.99）.The standard curve showed that 26 copies target genes per reaction could be detected by this method.The lowest detection limit of this method was 2 copies per μl.The method showed high species specificity and good reproducibility.Conclusion These results suggested that the Real-time PCR with TaqMan-MGB assay is highly specific and sensitive for the detection of O.tsutsugamushi,which might be applied for the diagnosis of this infection.