A method for the rapid screening of human blood samples for the presence of HIV-1 sequences: the probe-shift assay.
In theory, screening directly for the human immunodeficiency virus (HIV) DNA has advantages over screening for HIV-related antibodies; however, the HIV genome appears to be present only in a small fraction of peripheral blood cells from infected individuals. The polymerase chain reaction (PCR) specifically amplifies defined DNA segments, permitting the identification of specific DNA segments even if they are present in a fraction of cells. HIV-derived PCR-amplified segments have been detected by Southern transfer, "dot-blot" hybridization, and digestion with restriction endonucleases. Unfortunately, PCR may amplify not only the single segment of interest but also irrelevant segments, which can be incorrectly identified as HIV related. We describe here an alternative approach to specific detection of PCR-amplified DNA segments, the probe-shift assay, that is simpler, faster, and less subject to erroneous identification than the previously described techniques. The probe-shift assay relies on the hybridization of an appropriately labeled oligonucleotide probe to the amplified segment in solution. The hemiduplex formed between the amplified DNA and the probe is detected following fractionation on nondenaturing gels. This procedure is sufficiently sensitive to detect a single infected cell in the presence of more than 10(5) uninfected cells and has been applied directly to clinical samples.
Kumar
R
R
NCI-Frederick Cancer Research Facility, BRI-Basic Research Program, MD 21701.
Goedert
J J
JJ
Hughes
S H
SH
D000163:Acquired Immunodeficiency Syndrome / Q000097:blood* / Q000175:diagnosis; D001483:Base Sequence; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D005784:Gene Amplification; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008214:Lymphocytes / Q000382:microbiology; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification
D015342:DNA Probes; D004279:DNA, Viral