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目的建立一种敏感、特异的实时荧光定量PCR方法,用于检测临床标本中的恙虫病东方体。方法根据恙虫病东方体GroEL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。并从灵敏度、特异度、重复性及临床标本的检测能力等方面对本方法进行综合评价。结果建立的实时荧光定量PCR方法具有良好的特异性,可检测出恙虫病东方体Gilliam、Karp、Kato和Kawasaki株。建立的标准曲线循环阈值(Ct)与模板拷贝数呈现良好的线性关系(r=0.99),灵敏度分析显示样品最低检出浓度为21.8拷贝/μL。批内及批间重复性实验变异系数均小于1.5%,显示本方法具有良好的重复性。对本实验室保存的82份临床标本进行检测,并与常规巢式PCR方法比较,本方法检测出26份阳性标本中的25份,检出率为96.15%。结论本研究建立的实时荧光定量PCR方法具有较高的敏感性、特异性和稳定性,可用于临床病人及暴发疫情的实验室快速检测。
Objective To establish a sensitive and specific real-time fluorescence quantitative PCR method for the detection of Orientia tsutsugamushi in clinical specimens. Methods Primers and probes were designed according to the GroEL gene sequence of Orientia tsutsugamushi, and real-time PCR method was established. The method was evaluated comprehensively in terms of sensitivity, specificity, repeatability and detection ability of clinical specimens. Results The established real-time PCR method had good specificity and could detect Gilliam, Karp, Kato and Kawasaki strains of Orientia tsutsugamushi. The established standard curve showed a good linearity (r = 0.99) between the cycle threshold (Ct) and the copy number of the template, and the sensitivity analysis showed that the lowest detectable concentration was 21.8 copies / μL. The coefficient of variation of intra-and inter-batch reproducibility experiments were less than 1.5%, indicating that this method has good repeatability. 82 clinical specimens preserved in our laboratory were detected and compared with the conventional nested PCR method, 25 of 26 positive specimens were detected by this method and the detection rate was 96.15%. Conclusion The real-time fluorescence quantitative PCR method established in this study has high sensitivity, specificity and stability and can be used in clinical laboratory and rapid detection of outbreaks in laboratories.