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

为了在基因组水平上鉴定人巨细胞病毒（HCMV）的潜伏状态、再激活和产性感染，建立了聚合酶链反应（PCR）和联合逆转录-PCR （RT-PCR）方法。设计了两对编码LA和IE区的人工合成寡核苷酸引物，分别扩增HCMV AD169株Hind III-V片段305碱基对和Hind III-E片段346碱基Paris片段。PCR按照Saiki et al（1985）描述的程序进行。利用逆转录酶从RNA合成cDNA，进行RT-PCR。扩增产物通过凝胶电泳和用^<32> p标记的寡核苷酸探针进行点杂交检测。引物对HCMV株具有特异性，不能扩增其他疱疹家族病毒或人类基因组DNA。重组实验表明，用PCR方法可以检测到克隆扩增的HCMV DNA的10^<-5>pg。在感染巨细胞病毒72小时后，在MRC-5细胞中通过RT-PCR检测到IE mRNA。利用LA区编码对引物进行PCR检测是一种快速、灵敏的检测HCMV的方法，适用于尿、母乳等多种临床标本。因此，采用不同特异性引物的PCR和RT-PCR联合使用可能为诊断不同阶段的HCMV感染提供有用的工具。

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.