实时荧光定量PCR检测贝氏柯克斯体方法的建立

Quantitative detection of Coxiella burneti by real-time PCR

目的建立检测贝氏柯克斯体的Real-Time PCR方法。方法根据贝氏柯克斯体特有的htpAB基因相关重复序列(IS1111a)进行PCR扩增片断485bp,以克隆的IS1111a基因片断作标准DNA模板,建立Real-Time PCR检测方法。结果建立的Real-Time 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);重复性测试Ct变异系数CV≤6.1%;其灵敏性约为普通PCR的10倍;以其它相关立克次体和病原菌DNA为模板应用于该方法,结果均为阴性。结论所建立的Real-Time PCR方法具有很好的特异性、灵敏性和重复性,可代替普通PCR用于贝氏柯克斯体的感染早期的快速定性、定量检测。

Objective To establish real-time PCR for detection of Coxiella burneti.Methods A 485-bp fragment of Coxiella burneti.was amplified according to the htpAB-associated repetitive element（ISlllla） of coxiella burneti,cloned the fragment of ISlllla gene as the target sequence of amplification in real-time PCR assay.Results A linear relationship between threshold cycle（Ct） of the real-time PCR and the copy number was observed（r=0.999）,the coefficient of variation was CV≤6.1% in real-time PCR assay;its sensitivity was about 10 times higher than that of the general PCR in detection the homologous DNA assay;the results of detection of the DNA from other rickettsial agents and some pathogenic bacteria was negative.Conclusion The real-time PCR method established is highly specific,sensitive and reproducible and it can replace the general PCR technique in detection of coxiella burneti.