Development of Polymerase Chain Reaction Method for Detection of Human Cytomegalovirus DNA and Its Clinical Application.

为了从基因组水平上鉴定人巨细胞病毒（HCMV）的潜伏状态、再活化和生产性感染，建立了PCR和RT-PCR联合检测方法。设计了两对分别编码HCMV AD 169株DNA的LA和IE区的寡核苷酸引物，从Hind Ⅲ-V片段中扩增出305 bp的片段，从Hind Ⅲ-E片段中扩增出346 bp的巴黎片段。根据Saiki等（1985）描述的方法进行PCR。通过逆转录酶从RNA合成cDNA以进行RT-PCR。扩增产物通过凝胶电泳和斑点杂交与一个13 <32>P标记的寡核苷酸探针检测。该引物对HCMV株具有特异性，不扩增其他疱疹病毒家族病毒或人类基因组DNA。重组实验表明<-5>，PCR法可检测到10 μ g的HCMV DNA。用CMV感染MRC-5细胞72小时后，RT-PCR检测到IE mRNA。用一对编码HCMV LA区的引物进行PCR检测，可快速、敏感地检测尿液和乳汁中的HCMV，因此，采用不同特异性引物的PCR和RT-PCR联合应用，可为HCMV感染的不同阶段提供有用的诊断工具。

In order to identify latent state, reactivation and productive infection of human cytomegalovirus (HCMV) at genomic level, polymerase chain reaction (PCR) method and combined reverse transcription-PCR (RT-PCR) method were developed. Two pairs of synthetic oligonucleotide primers coding LA and IE region were designed to amplify the fragment of 305 base pairs from Hind III-V fragment or the fragment of 346 base Paris from Hind III-E fragment of HCMV AD169 strain DNA. PCR was carried out according to the procedure described by Saiki et al (1985). cDNA was synthesized from RNA by reverse transcriptase for performing RT-PCR. Amplified products were detected by gel electrophoresis and by dot blot hybridization with a ^<32>P-labeled oligonucleotide probe. The primers were specific for HCMV strains and did not amplify other herpes family viruses nor human genomic DNA. Reconstitution expriments demonstrated that 10^<-5>pg of the cloned amplified HCMV DNA was detectable by PCR method. IE mRNA was detectable in MRC-5 cells by RT-PCR until 72 hrs after infection with CMV. PCR using a pair of primers coding LA region was proved to be a rapid and sensitive method for detecting HCMV in various clinical specimens including urine and breast milk.Thus, a combined use of PCR and RT-PCR employing primers of different specificity might provide a useful tool for diagnosing various stages of HCMV infection.