Document 1270
 DOCN  M9591270
 TI    Coamplification of HIV type 1 and beta-globin gene DNA sequences in a
       nonisotopic polymerase chain reaction assay to control for amplification
       efficiency.
 DT    9509
 AU    Coutlee F; He Y; Saint-Antoine P; Olivier C; Kessous A; Department of
       Microbiology and Infectious Diseases, Notre-Dame; Hospital, Montreal,
       Quebec, Canada.
 SO    AIDS Res Hum Retroviruses. 1995 Mar;11(3):363-71. Unique Identifier :
       AIDSLINE MED/95306140
 AB    The polymerase chain reaction (PCR) fails to detect HIV-1 sequences in
       5% of infected individuals. To screen for false-negative PCR tests, we
       developed a nonisotopic PCR assay in which sequences from the
       beta-globin gene and from the HIV-1 vpu-env region were coamplified with
       biotinylated and fluorescein-labeled primers, respectively. Coamplified
       products were reacted with specific internal digoxigenin-labeled RNA
       probes. Hybrids were detected in a microtiter plate coated with
       streptavidin or anti-fluorescein antibody, with enzyme-labeled
       anti-digoxigenin antibody. After the optimization of the coamplification
       conditions, the assay could detect 5 proviral DNA copies in a lysate
       from 100,000 peripheral blood mononuclear cells. Fifty-seven samples
       from 55 HIV-1-seropositive patients and 25 samples from 25 seronegative
       individuals were evaluated. Fifty-two samples from HIV-infected
       individuals were positive for HIV-1 vpu-env sequences. Three of the 5
       PBMC lysates falsely negative for HIV-1 sequences had reactivities for
       beta-globin (3-23 fu) below that of 100,000 cells (304 fu). Nonisotopic
       coamplification allowed for the evaluation of the quality of specimens
       for PCR concurrently with the detection of the presence of viral
       template sequences.
 DE    Acquired Immunodeficiency Syndrome/BLOOD/*DIAGNOSIS  Base Sequence
       Comparative Study  DNA/*BLOOD  DNA Primers  DNA, Viral/*BLOOD  False
       Negative Reactions  Genes, env  Genes, vpu  Globin/*GENETICS  Human  HIV
       Infections/BLOOD/*DIAGNOSIS  HIV Seropositivity/BLOOD/*DIAGNOSIS
       HIV-1/GENETICS/*ISOLATION & PURIF  Immunoenzyme Techniques
       Lymphocytes/PHYSIOLOGY/VIROLOGY  Molecular Sequence Data  Polymerase
       Chain Reaction/*METHODS  Reproducibility of Results  RNA Probes  JOURNAL
       ARTICLE

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