聚合酶链反应检测致瘤性人类疱疹病毒6型方法的建立及应用

目的　建立致瘤性人类疱疹病毒 6型 (HHV 6 )荧光定量聚合酶链反应 (FQ PCR)检测方法 ,为快速、准确地定量检测HHV 6奠定基础。方法　根据HHV 6型基因序列 ,设计并合成引物、荧光标记探针。先用特异性引物筛选HHV 6 ,其PCR片段克隆入载体 ,再对重组质粒进行筛选、测序及鉴定。确证后的HHV 6DNA片段制成标准品并作为阳性模板 ,开发HHV 6FQ PCR检测系统。用该系统测定研究样品。结果　重组质粒获得的HHV 6DNA产物经 1∶10梯度稀释后 ,制作定量曲线的循环阈值 (Ct)与模板浓度具有良好的线性关系 ,相关系数为 0 99。经本反应系统检测的临床标本共135份 ,检测到 16份HHV 6DNA阳性。结论　建立了检测致瘤性HHV 6的FQ-PCR方法 ;此方法较定性PCR技术更为简便、快速、准确 ,且具有很好的应用前景和推广价值

Establishment and application of fluorescent quantitative PCR method for detecting human herpes virus type 6

OBJECTIVE: To establish fluorescent quantitative PCR method for detecting human herpes virus type 6 (HHV6). METHODS: According to the specific sequence of human herpes virus type 6 genes, the primers and the fluorescent probe (TaqMan) were designed and synthesized. The fragment generated from HHV 6 gene as template was cloned into the pMD18-T vector which was constructed from the pUC 18. And the positive recombinant plasmid was 1:10 diluted and used as standard quantitative template to make the standard curve for sample detection. RESULTS: The standard curve indicated the linear relationship between Ct (cycle threshold) and template concentration. The clinical samples from 135 cases were detected by this system, 16 cases among 135 were positive. CONCLUSION: The fluorescent quantitative PCR method for the detection of human herpes virus type 6 is simple and accurate, and this method may be helpful to clinical diagnosis.