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Poly A-linked non-isotopic microtiter plate reverse transcriptase assay for sensitive detection of clinical human immunodeficiency virus isolates.
Suzuki K; Saito T; Kondo M; Osanai M; Watanabe S; Kano T; Kano K; Imai
August 30, 1996
J Virol Methods. 1995 Nov;55(3):347-56. Unique Identifier : AIDSLINE

A colorimetric reverse transcriptase assay (cRT assay) was developed for quantitative detection of HIV-1. In this format, reverse transcriptase incorporates biotin-labeled dUTP onto oligo-dT primers hybridized to poly A templates. The templates are covalently bound to the surface of microtiter wells. The amount of incorporated biotin-labeled dUTP is measured by binding horseradish peroxidase conjugated streptavidin, washing away unbound peroxidase, adding colorimetric substrate and then reading with a standard colorimetric reader. The sensitivity of the assay is very good. As little as 3 x 10(5) molecules of recombinant HIV-RT can be detected after 20 h of reaction time. Direct comparison using 3 cultured clinical isolates indicates that this level of detection is equivalent to the commercially available p24 antigen capture assay and the HIV-RNA assay based on branched DNA signal amplification. Other retroviruses, such as HIV-2 and feline immunodeficiency virus (FIV), can also be detected in this format. This non-isotopic assay is easy to perform and could provide a convenient and quantitative method for HIV study by monitoring reverse transcriptase, an essential activity in the infection process.

Animal Antigens, Viral/ANALYSIS Colorimetry Comparative Study Evaluation Studies Human HIV/ENZYMOLOGY/ISOLATION & PURIF HIV Core Protein p24/ANALYSIS HIV-1/ENZYMOLOGY/*ISOLATION & PURIF HIV-2/ENZYMOLOGY/*ISOLATION & PURIF Immunodeficiency Virus, Feline/ENZYMOLOGY/ISOLATION & PURIF *Immunoenzyme Techniques Poly A Reagent Kits, Diagnostic RNA-Directed DNA Polymerase/*ANALYSIS RNA, Viral/ANALYSIS Sensitivity and Specificity Time Factors JOURNAL ARTICLE