输血传播病毒PCR-DHPLC检测技术的建立及初步应用

Development and preliminary application of PCR-DHPLC assays for detection of the Torque Teno virus

目的应用聚合酶链式反应(PCR)结合变性高效液相色谱(DHPLC)技术检测输血传播病毒。方法根据输血传播病毒的核酸序列特点设计特异性引物进行PCR,将扩增产物进行DHPLC检测分析,以验证方法的灵敏度、特异性、重复性,同时进行临床检测的初步应用。结果以乙型肝炎病毒、丙型肝炎病毒、戊型肝炎病毒进行特异性试验,无交叉反应,具有较好的特异性;重复性良好;灵敏度可达1.0×10~1拷贝/反应。分别应用实时荧光PCR法、普通PCR法及本文所建立的PCR-DHPLC法同时检测32份血清样本,发现有17份样本实时荧光PCR阳性,17份样本PCR-DHPLC阳性,15份普通PCR阳性。结论本文建立的PCR-DHPLC法具有特异、敏感、重复性好等优点,可用于输血传播病毒感染的分子流行病学调查。

In order to identify the Torque Teno virus(TT virus),a PCR-DHPLC assay was performed in this study.Primers specific were selected according to the characteristics of TT virus nucleic acid sequence to conduct PCR,and PCR products assayed by DHPLC.We analyzed the sensitivity,specificity,repeatability of PCR-DHPLC and applied it preliminarily on clinical detection.The specific testing was performed with TTV,HBV,HCV and HEV,no cross reaction were found,and the PCR-DHPLC assays we developed had good specification and nice repeatability.Sensitivity analysis showed that the developed PCR-DHPLC assays could detect 1.0×10~1 copy/μL.Then we detected 32 serum samples by this method,real-time PCR and normal PCR at same time.The results showed that 17 TTV positives results could be observed by PCR-DHPLC for 32 samples,it is consistent with real-time PCR test results and 15 positive by normal RT-PCR.PCR-DHPLC assays showed nice specification,sensitivity,repeatability,and could be used in epidemiological investigation.