Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: Optimization, validation, and comparative analysis with other detection systems

Shinji Yamamoto, Thomas M. Folks, Walid Heneine

    Research output: Contribution to journalArticlepeer-review

    26 Scopus citations

    Abstract

    An ultra-sensitive assay for reverse transcriptase (RT) activity called Amp-RT has been developed. An in vitro transcribed heteropolymeric RNA 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 Mn2+ concentrations using the human immunodeficiency virus type 1 (HIV-1) and the human T lymphotropic virus type 1 (HTLV-I), respectively, showed optimal assay reactivity for both viruses at 2-20 mM of Mg2+. Analysis of density banded HIV-1 showed that the peak RT activity of the assay was associated with the fractions consistent with retrovirus particles. The sensitivity of Amp-RT was also compared with that of three conventional RT assays by using seven different retroviruses including HIV-1, simian immunodeficiency virus (SIV), caprine arthritis-encephalitis 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 could not be detected by the three conventional RT assays. Amp-RT was able to detect all these viruses in 101-103-fold dilutions. Similarly, Amp-RT was found to be 103-106-fold more sensitive than the other RT assays in detecting HIV-1, SIV, or CAEV. Culture supernatants from 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 therefore, provides a useful tool for quantitating RT and retroviruses. Amp-RT offers a sensitive generic tool for the qualitative and quantitative detection of known and unknown retroviruses.

    Original languageEnglish (US)
    Pages (from-to)135-143
    Number of pages9
    JournalJournal of Virological Methods
    Volume61
    Issue number1-2
    DOIs
    StatePublished - Sep 1996

    Keywords

    • Retrovirus
    • Reverse transcriptase detection and quantitation

    ASJC Scopus subject areas

    • Virology

    Fingerprint Dive into the research topics of 'Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: Optimization, validation, and comparative analysis with other detection systems'. Together they form a unique fingerprint.

    Cite this