Single-tube fluorescent product-enhanced reverse transcriptase assay with Ampliwax (STF-PERT) for retrovirus quantitation.
A TaqMan fluorescent probe-based product enhanced reverse transcriptase (RT) assay is described in which the RT and polymerase chain reaction (PCR) steps are set-up in a single tube, in two compartments separated by Ampliwax (designated as single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT)). This simplification of the two-step method resulted in increased assay reproducibility and handling efficiency while maintaining the sensitivity of the PERT assay (<10 virions). The STF-PERT assay can be used to quantitate low amounts of retrovirus in clinical and research materials and to evaluate retrovirus contamination in cell substrates and biological products in human use.
Sears
Johnna F
JF
Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892, USA.
Khan
Arifa S
AS
D000818:Animals; D009189:Avian Myeloblastosis Virus / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000706:statistics & numerical data; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D015342:DNA Probes; D005456:Fluorescent Dyes