TaqMan MGB探针检测HBV DNA C区双突变方法的建立

Detection of Double Mutation of Basal Core Promoter(BCP) of HBV by TaqMan MGB

目的建立简便、灵敏的HBV DNA C区序列中核心启动子(BCP)双突变的检测方法。方法采用Taq-Man MGB探针技术进行双突变的检测,首先根据双突变的序列设计FAM荧光标记的TaqMan MGB探针和配套的引物,对标准阳性对照和标准阴性对照进行扩增检测,确定灵敏度和特异性,然后对临床HBV DNA阳性血清进行扩增检测,并通过PCR产物直接测序验证所建立方法检测结果的准确性。结果该方法检测出HBVDNA BCP双突变序列的灵敏度为3×100拷贝/ml的模板,并且可以稳定地从1×103拷贝/ml标准阴性对照中检测出1×101拷贝/ml的标准阳性对照。结论该技术适用于临床检测血清中HBV DNA C区BCP双突变。

OBJECTIVE To establish a simple, sensitive method for detecting the double mutation of the basal core promoter （BCP） of HBV. METHODS FAM fluorescence-labeled TaqMan MGB and primers driving from the region containing the double mutation of BCP were designed for the real time PCR, then the standard positive control, standard negative control and HBV DNA were amplified and detected by the real time PCR. The results of detecting the double mutation of BCP were validated by the direct-sequencing analysis of PCR products. RESULTS The double mutation of BCP of HBV could be detected by the real time PCR. The sensitivity of the method was 3 ×10^0 copy templates and as fewas 1% of mutant among wild-type virus sequence were detected. CONCLUSIONS The method can be used to detect the double mutation of BCP of serum HBV DNA.