One-step fluorescent probe product-enhanced reverse transcriptase assay.
Recently developed PCR-based reverse transcriptase (RT) assays are useful in the detection of retroviruses since they are approximately a millionfold more sensitive than conventional RT assays. However, these assays are both labor- and time-intensive. The previously described product-enhanced reverse transcriptase (PERT) assay involves a two-step RT-PCR followed by detection and quantitation of PCR products by either Southern blot or enzyme-linked immunosorbent assay (ELISA). We have modified the PERT assay to be a one-step, fluorescent probe, PCR-based RT assay that can be completed from sample dilution to final quantitative assay results in approximately 5 h without loss of assay sensitivity or specificity. The assay has a dynamic range of 6 logs, and therefore, extensive sample dilution is not necessary for quantitation. This newly enhanced fluorescent PERT assay can play an important role in the high-throughput detection of retroviral infection and characterization of RT activity.
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486-0004, USA.
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Arnold
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Hepler
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Keller
D000818:Animals; D009189:Avian Myeloblastosis Virus / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D004796:Clinical Enzyme Tests / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D006801:Humans; D009052:Leukemia Virus, Murine / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012192:Retroviridae Infections / Q000175:diagnosis*; D015301:Retroviruses, Simian / Q000201:enzymology / Q000235:genetics; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology / Q000821:virology
D015342:DNA Probes; D005456:Fluorescent Dyes; D012194:RNA-Directed DNA Polymerase