Real-time PCR与常规PCR在Q热立克体检测中的比较

Comparison of detection of Coxiella burneti by real-time PCR and conventional PCR method

目的 比较与评价Real-time PCR与常规PCR,寻求适合于Q热患者早期诊断的方法。方法 应用Taq Man探针定量PCR、SYBR Green染料定量PCR、巢式PCR和普通PCR四种方法分别对T-A克隆的Q热立克次体基因htp AB片断进行灵敏性、特异性和重复性检测及评价。结果 Taq Man PCR方法的灵敏度分别为SYBR Green PCR、巢式PCR和普通PCR的10倍、10倍和100倍。重复性测试Taq Man法Ct变异系数CV：0.2%-3.0%,SYBR Green法的CV：0.3%-6.0%。Taq Man和SYBR Green PCR均耗时约1h,巢式PCR耗时约2.5h,普通PCR耗时约1.5h。四种方法检测25种相关病原菌结果均为阴性。结论 Taq Man探针定量PCR方法具有很好的特异性、灵敏性、重复性和可操作性,可用于Q热患者临床早期诊断。

Objective To compare the detection effect of Coxiella burned by the real-time PCR and conventional PCR for early diagnosis of Q fever patients. Methods T-A cloned the fragment of Coxiella burneti htpAB gene as the target sequence were amplified by real-time PCR assay（TaqMan PCR and SYBR Green PCR） and conventional PCR （nested PCR and general PCR ） . Results The sensitivity of TaqMan PCR was 10 times higher than that of SYBR Green PCR 10 times higher than that of nested PCR, and 100 times higher than that of general PCR. The coefficients of Ct variation （CV） of TaqMan PCR and SYBR Green PCR were 0.2%-3.0% and 0.3%-6.0%, respectively. It took about an hour to complete a test for both TaqMan and SYBR Green PCR, about 2.5 hours for nested PCR, and about 1.5 hours for general PCR. The results of detection of DNA of 25 species of DNA from rickettsia and other commen agents were all detected for negative by the four methods respectively. Conclusion These results showed that TaqMan PCR for deteting Coxiella burneti was the most specific, sensitive, repeating and operable in four methods, and it can be used for early clinical diagnosis of Q fever patients.