Poly A-linked non-isotopic microtiter plate reverse transcriptase assay for sensitive detection of clinical human immunodeficiency virus isolates.
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.
Suzuki
K
K
Biomedical Research Center, Olympus Optical Co. Ltd., Tokyo, Japan.
Saito
T
T
Kondo
M
M
Osanai
M
M
Watanabe
S
S
Kano
T
T
Kano
K
K
Imai
M
M
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D003124:Colorimetry; D005069:Evaluation Studies as Topic; D006678:HIV / Q000201:enzymology / Q000302:isolation & purification; D016655:HIV Core Protein p24 / Q000032:analysis; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology / Q000302:isolation & purification*; D015498:HIV-2 / Q000201:enzymology / Q000302:isolation & purification*; D006801:Humans; D016089:Immunodeficiency Virus, Feline / Q000201:enzymology / Q000302:isolation & purification; D007124:Immunoenzyme Techniques; D011061:Poly A; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013997:Time Factors
D000956:Antigens, Viral; D016655:HIV Core Protein p24; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D011061:Poly A; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase