猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。

Establishment of Nested PCR Method for Detection of Rhesus Cytomegalovirus (RhCMV) and Its Preliminary Application

Objective To establish PCR detective method of the laboratory monkeys and to use it preliminarily.Methods Two pairs of primers were designed according to the conserved sequence(Rh85) from rhesus cytomegalovirus 68-1 strain's in the GenBank,and HCMV positive control and 20 samples of rhesus monkey blood were detected with nested PCR.Among them,a positive PCR product was cloned and sequenced.Results The results indicated both HCMV positive control and 2 samples were positive.The amplified fragment of samples was high homology comparing with RhCMV Rh85 gene sequence in the GenBank by using BLAS software.Conclusion A sensitive,specifical method of nested PCR was established for detection of rhesus cytomegalovirus(RhCMV).