实时荧光定量PCR检测恙虫病东方体

Detection of Orientia tsutsugamushi by quantitative real-time PCR assay

目的建立检测恙虫病东方体的实时荧光定量PCR(quantitative real-ti me PCR)方法。方法根据恙虫病东方体56kD外膜蛋白基因序列设计引物和探针,以克隆的56kD基因片段作DNA模板,建立实时荧光定量PCR检测方法。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(r=0.999)。荧光定量PCR检测恙虫病东方体的灵敏度约为套式PCR的100倍,并且具有良好的重复性。用该定量PCR检测其它相关立克次体和病原菌DNA样本,检出结果均为0。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肺脏、肝脏标本,结果脾脏中东方体出现最早和检出量最多,肝脏和肺脏次之。血中的恙虫病东方体量较低。结论本研究建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于东方体感染早期血样本的快速检测作恙虫病感染早期诊断,并且可以定量分析评价恙虫病东方体感染的程度。

A pair of primers and a TaqMan probe were designed according to the 56 kDa-outer membrane gene sequence of Orientia tsutsugamushi in order to develop the quantitative real-time PCR assay. A linear relationship between threshold cycle （Ct） of the quantitative real-time PCR and the DNA copy number could be demonstrated （r= 0. 999）. The sensitivity of this method of assay to detect the homologous DNA was about 100 times higher than that of the nested PCR, with high species-specificity and good reproducibility. This method was successfully employed in the detection of O. tsutsugamushi in different samples, blood and tissues of liver, spleen, and lungs of the infected mice. However, the detection of the DNA from other rickettsial agents and some of the pathogenic bacteria appeared to be negative. These results suggest that the quantitative real-time PCR assay is highly specific and sensitive for the detection of O. tsutsugamushi, and it may be used for the rapid diagnosis of this infection.