实时定量HSVPCR实验中一种内参的建立

A novel real-time PCR method with internal controls for HSV

目的建立一个内参用于单纯疱疹病毒(Herpes simplex virus,HSV)HSV1、HSV2的PCR诊断实验。方法用HSV的质粒嵌合引物两步PCR扩增质粒DNA和HSV的引物获得HSVPCR诊断实验的内参。结果内参混合到含8份HSV1和9份HSV2(总共17份)的阳性组织培养中,发现内参对靶序列没有抑制或很少抑制。对272份临床标本加入内参或没有内参扩增的结果有非常好的一致性。结论该内参可以用于常规PCR检测HSV,对于发现实验过程是否存在抑制物具有指示意义。

Objective The study＇s aim is to develop an internal control for clinical routine diagnostic HSV 1 and 2 PCR assay that has identical primer binding sites to the HSV target DNA but an internal sequence derived from plasmid vector pGEM-T and detected by a different probe and fluorophor.Methods Production of the internal control was achieved using a straightforward two-step PCR technique in which plasmid DNA was amplified with HSV-plasmid chimeric primers,followed by amplification of the resulting amplicons with HSV primers and purification for later use.Results Both the internal control and viral DNA were amplified in initial tests with 8 samples of tissue-culture HSV 1 and 9 samples of HSV2 positives（17 in total）,with little or no inhibition of the target sequences.A high level（98%） of concordant results was obtained with 272 clinical samples with herpetic lips assayed in parallel with and without the internal control.Conclusion These results are sufficient to justify the incorporation of the internal control into the routine HSV DNA assay in our laboratory.