建立染料法荧光定量PCR检测恙虫病东方体

目的建立染料法荧光定量PCR检测恙虫病东方体。方法根据恙虫病东方体56kD外膜蛋白基因序列设计特异性引物,建立染料法(SYBR green I)实时荧光定量PCR,评估其灵敏性及特异性;并对恙虫病东方体鸡胚培养物、东方体感染小白鼠脏器标本以及恙虫病病人血样等多种样本进行检测。结果建立的荧光定量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(r2=0.99),灵敏性评估显示20μl体系单PCR反应管靶基因大于28个拷贝即可被有效检测,最低检出浓度为2拷贝/μl,比普通PCR检测方法提高1~2个数量级,并且具有较好的重复性;建立的SYBR I染料法具有良好的特异性,与立氏立克次体R株等其他5种立克次体,以及被检测的其他几种病原菌DNA模板均不发生特异性扩增;用建立的染料法对东方体鸡胚培养物、东方体感染动物脏器,以及采集的现场恙虫病病人血样共59份标本进行检测,其中57份检出阳性。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肾脏、肝脏标本,结果脾脏中东方体检出量最多,肝脏和肾脏次之,血中的恙虫病东方体量较低。结论本研究建立荧光定量PCR方法均具有很高的特异性和敏感性,适合各种样本中东方体的快速检测,可用于实验室的快速诊断。

Detection of Orientia tsutsugamushi by dye quantitative real-time PCR assay

Objective To establish dye real - time PCR method for the rapid detection of Oreintia tsutsugamuzhi （Ot）. Methods A pair of specific primers were designed according to the 56kD outer membrane protein gene se- quence of Oreintia tsutsugamushi in order to establish the dye method （SYBR green I） real-time quantitative PCR and its sensitivity and specificity were assessed. Various samples including standard Ot strains from chick embryo cultures, Ot isolates from animal organs and patients＇ blood were detected. Results A linear relationship between threshold cycle （Ct） of the quantitative real-time PCR and the DNA copy number could be demonstrated （ r2 = 0.99）. Sensitiv- ity assessment found that only 28 copies of the gene can be detected in each 20 p,1 PCR reaction tube,and the mini- mum detectable concentration was 2 copies/p,1, presenting an increase by 2 orders of magnitude than the ordinary PCR and having good repeatability ;The SYBR I dye method has good specificity for the other five kinds of Rickettsia and other pathogenic DNA templates presenting no specific amplification ;57 of the total 59 samples including the standard Ot strains from chick embryo cultures and Ot isolates from animal organs and patients＇ blood were detected positive. This method was also successfully employed to compare the quantity of Orientia tsutsugamushi in different organs suchas blood, liver, spleen and kidney of the infected mice resulting that the most in spleen, then in liver and kidney, and the least in blood. Conclusion These results suggest that the dye quantitative real-time PCR assay presents highly specific and sensitive and is suitable for the detection of Orientia tsutsugamushi in various samples, so it may be em- ployed successfully in the rapid diagnosis scrub typhus