| Project/Area Number |
01480261
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pediatrics
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| Research Institution | Sapporo Medical College |
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Principal Investigator |
CHIBA Shunzo Sapporo Medical College, Department of Pediatrics, Professor, 医学部, 教授 (50045374)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Nobuhiro Sapporo Medical College, Department of Pediatrics, Instructor, 医学部, 助手 (50216420)
YOSHIDA Koichi Sapporo Medical College, Cancer Institute, Assistant Prof., がん研究所, 講師 (60117653)
YAMANAKA Tatsuru Sapporo Medical College, Department of Pediatrics. Associate Prof., 医学部, 助教授 (70045508)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1990: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | Human cytomegalovirus / PCR / RT-PCR / DNA hybridization / RTーPCR法 / ドット・ブロット・ハイブリダイゼ-ション |
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| Research Abstract |
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.
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