Analysis of Human Immunodeficiency Virus Type 1 Integration by Using A Specific, Sensitive and Quantitative Assay Based on Real-time Polymerase Chain Reaction
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A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA sysnthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.
KeywordsHIV-1 Integration Alu-PCR Real-time PCR Nested PCR
We thank Dr. Naoki Yamamoto for his support and helpful discussions. This work was supported by grants from the Ministry of Education, Science and Culture and the Ministry of Health, Labor and Welfare of Japan.
- 19.S.L. Butler, M.S. Hansen, F.D. Bushman, Natl. Med. 7, 631–634 (2001)Google Scholar