인간 세포거대바이러스의 배양상 및 PCR에 의한 바이러스 DNA 검색

Abstract

In order to understand the cultural characteristics of human cytomegalovirus (HCMV) and to make direct detection of viral DNA during cell culture, the viruses were cultured in MRC-5 fibroblast cell line and culture supernatants were tested by the Polymerase Chain Reaction (PCR). Charactistic cytopathic effects (CPE) of HCMV infection were detected after 10 days of virus inoculation. Characteristic intranuclear inclusion bodies and large, round or oval shaped cells were clearly seen after 12 days of virus inoculation. In PCR, oligonucleotide pairs for major immediate early (MIE) gene of HCMV were used as primers. Amplified products were detected by gel electrophoresis and by Southern blot hybridization with digoxigenin-labeled HCMV MIE gene probe. In estimating the sensitivity of PCR, the PCR product starting with l/i\ aliquot of an infectivity titer of 102 5 TCIDso/0.2ml of cell culture mixture was detedcted by direct agarose gel electrophoresis and Southern blot hybridization. The specificity of the PCR was evaluated using other members of the herpes family of viruses and various DNAs. No amplification was noted by direct gel analysis or by Southern blot hybridization and only HCMV containing specimen was positive. In the sensitivities and the specificities, there were no differences between the PCR with OPC(oligonucleotide purifica¬tion column) purified primers and the PCR with Speed Vac dried primers (no other purification steps were used). Culture supernatants were tested for the detection of MIE DNA by PCR. After 4 days of cul¬ture, the MIE DNA was detected in culture supernatants by PCR. In conclusion, I think we can make more rapid and precise diagnosis of HCMV infection by combining virus culture and PCR of culture supernatants. Key Words： Human Cytomegalovirus (HCMV), Culture, Major Immediate Early gene (MIE), PCR.