Detection of HIV‐1 DNA in Crude Cell Lysates of Peripheral Blood Mononuclear Cells by the Polymerase Chain Reaction and Nonradioactive Oligonucleotide Probes
作者:
B. Conway,
K. Adler,
L. Bechtel,
J. Kaplan,
M. Hirsch,
期刊:
Journal of Acquired Immune Deficiency Syndromes
(OVID Available online 1990)
卷期:
Volume 3,
issue 11
页码: 1059-1064
ISSN:0894-9255
年代: 1990
出版商: OVID
关键词: PCR;Cell lysates;Alkaline phosphatase probe.
数据来源: OVID
摘要:
SummaryThe 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-1polgene. Bracket and nested amplification protocols were used. Products were identified by dot-blot hybridization or agarose gel eleclrophoresis 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.
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