A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation.
A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibody and colorometric reading. The system also allows the use of variable amounts of primer. The kinetics and characteristics of the assay using BrdUTP is similar to the use of [3H]dTTP. The sensitivity of the assay can be varied either by altering the duration of RT assay time and/or by prolonging the alkaline phosphatase reaction. Thus the assay can detect < 0.02 pg of recombinant human-immunodeficiency-virus (HIV) type I RT, < 0.005 m unit of avian-myeloblastosis-virus RT or < 0.02 m unit of recombinant Moloney-murine-leukaemia-virus RT. The assay was found to be useful with various types of cell-culture material, and a comparative study of 16 HIV-infected lymphocyte cultures, using 10 microliters of supernatant medium for RT assay and 22.5 microliters for p24 antigen assay showed that the new RT assay was at least 25-fold more sensitive than the p24 antigen assay. The results also show a good correlation between the RT activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the RT assay. The technical performance and the capacity of the test compared with other available RT kits is discussed, as well as its use for other applications.
Ekstrand
D H
DH
Department of Medical Genetics, Uppsala University, Sweden.
Awad
R J
RJ
Källander
C F
CF
Gronowitz
J S
JS
D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D003856:Deoxyuracil Nucleotides; D006678:HIV / Q000302:isolation & purification*; D015658:HIV Infections / Q000201:enzymology; D006801:Humans; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000201:enzymology; D013698:Templates, Genetic; D013942:Thymine Nucleotides; D017186:Titrimetry
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; C032549:bromodeoxyuridine triphosphate; D004247:DNA; D012194:RNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate