Document 0277
 DOCN  M9590277
 TI    Development of a quantitative HIV-1 RNA PCR assay.
 DT    9509
 AU    Dowton DN; Dwyer DE; Cunningham AL; Department of Virology, ICPMR,
       Westmead Hospital.
 SO    Annu Conf Australas Soc HIV Med. 1994 Nov 3-6;6:245 (unnumbered poster).
       Unique Identifier : AIDSLINE ASHM6/95291822
 AB    The aim of this project is to develop an assay to quantitate the levels
       of circulating HIV-1 RNA in plasma of infected patients, and to apply
       this assay to antiretroviral drug trials and other clinical situations.
       Duplicates of plasma are spun by ultracentrifugation and lysed using a
       guanidium thiocyanate containing buffer (Chomczynski et al, 1986). A
       first round PCR is performed on the liberated RNA using primers located
       in the pol gene of the virus. A second round (nested) PCR is then
       performed on the first round product using internal primers, one of
       which is biotinylated, as well as 35S-dCTP. This yields a biotinylated,
       35S labelled product, which is then captured on the wells of a
       microtitre plate previously coated with streptavidin. Following
       extensive washing, the intensity of the captured product is determined
       by liquid scintillation counting. To quantitate, the counts obtained
       from the patient's specimens are compared to a standard curve generated
       by performing nested PCR on specimens of known copy number. These
       standards were generated by in vitro transcription of PCR product which
       has been flanked with the sequence for the T7 RNA polymerase promoter.
       By this method, specimens containing RNA copy numbers in the range of 10
       to 10 x 10(6) copies/ml can be quantitated. Applications of this assay
       include drug trials, disease progression studies and assessment of body
       fluids (eg saliva, semen).
 DE    Antiviral Agents/THERAPEUTIC USE  Clinical Trials  Human  HIV
       Infections/*DIAGNOSIS/DRUG THERAPY  *HIV-1/GENETICS  Polymerase Chain
       Reaction/*METHODS  RNA, Messenger/*BLOOD/DRUG EFFECTS  MEETING ABSTRACT

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