用双重PCR检测人巨细胞病毒DNA

Detection of cytomegalovirus DNA with double PCR

选取HCMV IEV基因区为扩增的靶序列,合成两对引物,其中一对巢居于另一对之间。扩增片段分别为721 bP和167bp。单次PCR扩增灵敏度达0.5～1.0fg,双重扩增可提高灵敏度10～100倍。用HSV-1,HSV-2,CoxB组1～6型病毒培养物作模板,无非特异扩增。14例疑为CMV感染患儿尿标本经PCR测定,12例检出HCMV DNA,其中11例病毒分离阳性。PCR快速,可靠,比病毒分离简便易行,对临床HCMV感染的诊断有重要意义。

A double PCR assay was developed to amplify conserved sequences from the immediate early gone of human cytomegalovirus(HCMV).The first primer set was selected to amplify a 721 bp fragment and the next primer set was nested within the first and amplified a 167 bp fragment.With single PCR system 0.5fg purified HCMV DNA was detectable but the sensitivity was increased 10～100 times in double PCR system.When cell cultures infected with HSV-1,HSV-2,and coxsackio B viruses served as templates,no positive results were found.14 urine samples from children with suspected HCMV infection were analysed by single PCR,double PCR and virus cultivation.The double PCR was the most sensitive method.This suggests that detection of HCMV by double PCR is rapid,more sensitive and simple in performance than by virus isolation,it is of great importance in diagnosis of HCMV infections.