6th International AIDS Conference


San Francisco, California, USA — June 20-23, 1990

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Detection of HIV by a non-radioactive sensitive reverse transcriptase assay.

Int Conf AIDS 1990 Jun 20-23; 6:321 (abstract no. 1028)
Lee M, Kusakabe H, Reisinger D, Takasaki T, Sano K, Imagawa D; Harbor-UCLA Medical Center, California, USA

OBJECTIVE: To develop a reverse transcriptase (RT) assay that does not require use of a radioisotope, we tested a dTTP analog, digoxigenin-dUTP (Boehringer Mannheim Biochemicals), for its ability to be made into DNA.

METHODS: The in vitro RT assay employed a poly rA oligo dT synthetic template primer at 37 degrees C for 18 hours. The DNA product was collected by TCA precipitation and dot blotting onto nitrocellulose. Detection was by an anti-digoxigenin alkaline phosphatase conjugate and standard phosphatase assay.

RESULTS: Digoxigenin d-UTP was used by HIV RT to make a DNA product. The specificity was verified by the RT reaction occurring only on a poly rA oligo dT template. Under similar assay conditions, the Moloney murine leukemia virus RT was also active only on the poly rA oligo dT template, while E.coli DNA polymerase I was active only on a poly dA oligo dT template. This RT assay was able to detect as few as 250 HIV particles. In comparison, the assay employing 3H-dTTP detected 125 HIV particles, while the HIV p24 antigen capture-ELISA assay detected 75 HIV particles.

CONCLUSION: HIV can be detected in a non-radioactive RT assay that is slightly less sensitive than the presently used radioisotope assay. However, it offers an alternative to laboratories who may not wish to work with radioactive compounds and may also be used as a confirming test for a positive result by the HIV antigen capture assay.

Keywords: AEGIS, RNA-Directed DNA Polymerase, HIV, Templates, Genetic, Thymine Nucleotides, HIV Core Protein p24, Oligodeoxyribonucleotides, Poly T, Poly A, Reverse Transcriptase Inhibitors, HIV Infections, Digoxigenin, thymidine 5'-triphosphate, oligo (dT), poly A-T, poly(dA), In Vitro, genetics, ICA6

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Copyright © 1990 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.