title: Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection.
abstract: A method for quantitating human immunodeficiency virus type 1 plasma viremia may be useful in monitoring disease progression and the responsiveness of patients to a therapeutic regimen or vaccine. A quantitative assay for viral RNA in plasma or sera that differs in several aspects from those reported previously was developed. First, whereas conventional reverse transcriptase-PCR assays involve a two-step process and use two enzymes, the method described uses a single enzyme, rTth DNA polymerase, for both reverse transcription and PCR. The reactions are carried out in a single tube and with a single buffer solution with uninterrupted thermal cycling. Second, uracil-N-glycosylase and dUTP are incorporated into the reaction mixtures to ensure that any carryover of DNA from previous amplifications will not compromise quantitation. Third, a quantitation standard is incorporated into each reaction mixture so that differences in amplification efficiency caused by sample interferents, variability in reaction conditions, or thermal cycling can be normalized. To ensure comparable amplification efficiency, the quantitation standard has the same primer-binding regions as the human immunodeficiency virus type 1 target and generates an amplified product of the same size and base composition. The probe-binding region was replaced with a sequence that can be detected separately. Fourth, a colorimetric detection format was modified to provide at least a four-log-unit dynamic range. The quantitative assay requires only a single amplification of the sample and can be completed in less than 8 h. The procedure was used on archival samples to demonstrate the viremic spike in acute infection and the suppressed levels of circulating virus following seroconversion.
authors.affiliation: Department of Infectious Diseases, Roche Molecular Systems, Inc., Alameda, California 94501.
authors.forename: J
authors.initials: J
authors.lastname: Mulder
authors.forename: N
authors.initials: N
authors.lastname: McKinney
authors.forename: C
authors.initials: C
authors.lastname: Christopherson
authors.forename: J
authors.initials: J
authors.lastname: Sninsky
authors.forename: L
authors.initials: L
authors.lastname: Greenfield
authors.forename: S
authors.initials: S
authors.lastname: Kwok
mesh_terms: D000208:Acute Disease; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D015658:HIV Infections / Q000382:microbiology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics*; D015203:Reproducibility of Results; D014766:Viremia / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase