Abstract
We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non- radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 μg of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.
| Original language | English |
|---|---|
| Pages (from-to) | 235-240 |
| Number of pages | 6 |
| Journal | Molecular and Cellular Probes |
| Volume | 8 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 1 Jan 1994 |
| Externally published | Yes |
Keywords
- Digoxigenin-11- dUTP
- Human immunodeficiency virus
- Polymerase chain reaction
- Reverse-blot hybridization
