HIGHLY SENSITIVE QUALITATIVE AND QUANTITATIVE DETECTION OF REVERSE-TRANSCRIPTASE ACTIVITY - OPTIMIZATION, VALIDATION, AND COMPARATIVE-ANALYSIS WITH OTHER DETECTION SYSTEMS

An ultra-sensitive assay for reverse transcriptase (RT) activity calle d Amp-RT has been developed. An in vitro transcribed heteropolymeric R NA sequence was used as a template, and polymerase chain reaction (PCR ) amplification with Southern-blot hybridization served as a detection system for the cDNA product of the reaction. Titration of Mg2+ and Mn 2+ concentrations using the human immunodeficiency virus type 1 (HIV-1 ) and the human T lymphotropic virus type 1 (HTLV-I), respectively, sh owed optimal assay reactivity for both viruses at 2-20 mM of Mg2+. Ana lysis of density banded HIV-1 showed that the peak RT activity of the assay was associated with the fractions consistent with retrovirus par ticles. The sensitivity of Amp-RT was also compared with that of three conventional RT assays by using seven different retroviruses includin g HIV-1, simian immunodeficiency virus (SIV), caprine arthritis-enceph alitis virus (CAEV), HTLV-I and HTLV-II, simian retrovirus type 2 (SRV -2), and gibbon ape leukemia virus (GALV). HTLV-I, HTLV-II, and GALV c ould not be detected by the three conventional RT assays. Amp-RT was a ble to detect all these viruses in 10(1)-10(3)-fold dilutions. Similar ly, Amp-RT was found to be 10(3)-10(6)-fold more sensitive than the ot her RT assays in detecting HIV-1, SIV, or CAEV. Culture supernatants f rom uninfected cell lines were all Amp-RT negative. A quantitative Amp -RT assay was also developed by using recombinant HIV-1 RT and signal quantitation. The assay was found to have a 5 log linear range, and th erefore, provides a useful tool for quantitating RT and retroviruses. Amp-RT offers a sensitive generic tool for the qualitative and quantit ative detection of known and unknown retroviruses.