Detection of HIV-1 DNA in crude cell lysates of peripheral blood mononuclear cells by the polymerase chain reaction and nonradioactive oligonucleotide probes.
The aim of this study was to detect HIV-1 proviral DNA in lysates of peripheral blood mononuclear cells (PBMCs) by the polymerase chain reaction (PCR) and hybridization with a nonradioactive probe. PBMCs were lysed in 1% Triton X-100. PCR was then carried out using primers complementary to a conserved region of the HIV-1 pol gene. Bracket and nested amplification protocols were used. Products were identified by dot-blot hybridization or agarose gel electrophoresis and Southern hybridization, using an alkaline phosphatase-linked oligonucleotide probe specific for amplified sequences. Colorimetric and chemiluminescent substrates were used. HIV-1 DNA was detected in PBMCs of 57/59 HIV-1-seropositive individuals, 8 of which were positive only following the use of nested primers. Of 12 seropositive samples that were negative by other HIV-1 diagnostic tests (PBMC coculture and serum p24 antigen detection), 11 were positive by PCR. PCR using PBMC lysates is a very sensitive method of detecting HIV-1 proviral sequences. The use of nested primers appears to increase the sensitivity of the procedure.
Conway
B
B
Infectious Disease Unit, Massachusetts General Hospital, Boston 02114.
Adler
K E
KE
Bechtel
L J
LJ
Kaplan
J C
JC
Hirsch
M S
MS
D001483:Base Sequence; D015139:Blotting, Southern; D004279:DNA, Viral / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D015683:Gene Products, gag / Q000097:blood; D016655:HIV Core Protein p24; D006679:HIV Seropositivity / Q000382:microbiology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D007963:Leukocytes, Mononuclear / Q000382:microbiology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D014758:Viral Core Proteins / Q000097:blood
D004279:DNA, Viral; D015683:Gene Products, gag; D016655:HIV Core Protein p24; D015345:Oligonucleotide Probes; D014758:Viral Core Proteins