贝氏柯克斯体实时荧光定量PCR方法的建立及对云南鼠标本检测

Establishment of real-time PCR in detecting of Coxiella burnetii and its application to testing mouse samples collected from Yunnan province

目的建立贝氏柯克斯体荧光定量PCR方法并应用于云南省鼠标本的检测。方法根据贝氏柯克斯体ISlllla基因设计引物和探针,以克隆的ISlllla基因片断(485bp)作标准DNA模板,建立实时荧光定量PCR检测方法,并与普通巢式PCR方法进行比较。采用2种方法对云南玉溪自然界采集的鼠各类标本进行检测分析,计算2种方法的检出率。结果实时荧光定量PCR标准曲线的循环阈值(cycle threshold,Ct)与模板拷贝数呈良好的线性关系(r=-0.999),重复性测试Ct变异系数(coefficient ofvariation,CV)为0.25%～3.98%,灵敏度为巢式PCR的10倍。以其他立克次体和常见病原菌共26种DNA为模板进行特异性检测,结果均为阴性。实时荧光定量PCR检测鼠血、肝脏、脾脏中的贝氏柯克斯体,阳性率分别为32.31%、61.29%、85.19%;巢式PCR法检测相应标本阳性率分别为27.69%、11.29%、74.07%;间接免疫荧光试验检测鼠血清中贝氏柯克斯体IgG抗体阳性率为26.15%。结论本实验建立的实时荧光定量PCR比巢式PCR敏感,且具有良好的特异性,可用于人群和动物贝氏柯克斯体感染监测及临床标本的检测。

Objective To establish real-time quantitative PCR in detecting Coxiella bumetii and evaluate its application to testing mouse samples collected from Yunnan province. Methods Taqman-probe and primers were designed and a 485 bp fragment was amplified based on the btpAB repetitive element （Islllla） of Coxiella burneti and the recombinant plasmid containing the ISlllla gene was made as the standard templet, thus the real-time quantitative PCR was established. The established real-time quantitative PCR and the nested PCR were compared in detecting blood, liver and spleen samples taken from the mice of Yuxi city, Yunnan province. Detection rates of the two methods were calculated. Results There was a good linear correlation between cycle threshold（Ct） of the real-time quantitative PCR and the copy number of reference templets（r =-0.999）. Ct coefficient of variation of the real-time quantitative PCR in repeated experimental assays was between 0.25% and 3.98%. The sensitivity of the real-time quantitative PCR was 10 times higher than that of the nested PCR. The results of specificity detection of 26 species of Rickettsia and other common agents in clinics proved to be negative. The positive rates of Coxiella burnetii in the blood, liver and spleen samples of the mice by real-time quantitative PCR and the nested PCR were 32.31% vs 27.69%, 61.29% vs 11.29%, and 85.19% vs 74.07%, respectively. The positive rate of serological IgG antibody to Coxiella bumetii in the mice by indirect immunofluorescence assay was 26.15%. Conclusions With good specificity, the real-time quantitative PCR established in the study is more sensitive than the nested PCR. It can be used in the surveillance of rickettsial infection in humans and animals and in the detection of clinical samples as well.