Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates.
The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.
Sauvaigo
S
S
CIS Bio International, Gif sur Yvette, France.
Barlet
V
V
Guettari
N
N
Innocenti
P
P
Parmentier
F
F
Bastard
C
C
Seigneurin
J M
JM
Chermann
J C
JC
Teoule
R
R
Marchand
J
J
D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000382:microbiology; D004279:DNA, Viral / Q000097:blood / Q000235:genetics*; D005069:Evaluation Studies as Topic; D015750:Genes, gag; D006679:HIV Seropositivity / Q000175:diagnosis / Q000382:microbiology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D004279:DNA, Viral