随机扩增多态性技术联合荧光定量高敏检测三种疱疹病毒方法的建立

目的:建立一种随机扩增多态性技术(RAPD)联合荧光定量聚合酶链式反应(q PCR)高敏定量检测单纯疱疹病毒(HSV)、人类巨细胞病毒(HCMV)、水痘带状疱疹病毒(VZV)的新方法。方法:根据文献筛选出数十条随机引物,分别对三种疱疹病毒进行随机扩增,产物经2%琼脂糖凝胶电泳,选取稳定清晰条带进行分离、纯化及克隆测序,应用blast-nr比对genebank现有病毒序列,选取高于99%匹配度的基因序列作为目的片段,并在其内部用primer3.0设计特异性内引物。经过引物筛选及条件优化后建立RAPD联合q PCR检测新方法,分别检测三种疱疹病毒。结果:经过筛选,确定了HSV、HCMV、VZV扩增灵敏性及特异性最好一组引物,建立的RAPD-q PCR可分别检测出1:10~6 HSV、1:10~5 HCMV和1:10~5 VZVDNA,而单一q PCR仅能检测1:10~3 HSV、1:10~2HCMV和1:103 VZVDNA。RAPD-q PCR相比于单一q PCR灵敏性提高100-1000倍,RAPD-q PCR扩增大于1:10~5病毒DNA得到的CT值均小于22.96±0.81,与10~2 copies/μL的标准线相距远,易于区分阴性和阳性。此外,用乙型肝炎病毒及疱疹病毒互为对照未见非特异扩增,特异性好。结论:随机扩增多态性技术联合荧光定量是一种检测三种病毒高度灵敏及特异的检测方法。

Random Amplified Polymorphic DNA Combined with Real-time PCR for Detecting Herpes Virus

ABSTRACT Objective: To develop a more sensitive method for quantitative detection of herpes simplex virus, human cy- tomegalovirus,varicella-zoster virus by random amplified polymorphic DNA combined with fluorescence quantitative polymerase chain reaction (RAPD-qPCR). Methods: According to the literature, dozens of random primers for RAPD were selected and randomly ampli- fied three herpes viruses. The stable and clear bands were selected to isolate, purify and sequence from 2% agarose gel electrophoretic plate. Then more than 99% matched gene sequences were chosen as the target fragment compared with the genebank existing virus se- quences using blast-nr and the internal, specific primers were designed with primer 3.0. Finally, the new method of RAPD combined with qPCR afterprimer selection and reaction condition optimization was established and used to detect three herpes viruses. Results: After screening, a set of the best random primer and specific primer for each virus was chosen, and the RAPD-qPCR could detect 1: 10⁶6 HSV, 1: 10⁵ HCMV and 1: 10⁵ VZVDNA respectively, while the single qPCR could detect only 1: 10³ HSV, 1: 10² HCMV and 1: 10³ VZVD- NA. The sensitivity of RAPD -qPCR is higher 100-1000 times than qPCR. CT value was less than 22.96±0.81 when detecting more than 1:105 diluted virus DNA by RAPD -qPCR, so that the results was easy to distinguish. Conclusion: RAPD-qPCR was a more sensitive and specific method to detect three herpesviruses than single qPCR.