A sensitive nonisotopic hybridization assay for HIV-1 DNA.
We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA.
Keller
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GH
Biotech Research Laboratories, Inc., Rockville, Maryland 20850.
Huang
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DP
Manak
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MM
D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D015497:HIV-1 / Q000235:genetics*; D008722:Methods; D009693:Nucleic Acid Hybridization; D019914:Taq Polymerase
D015342:DNA Probes; D004279:DNA, Viral; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase