Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.
A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.
Murakami
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Process Development Department, Berlex Biosciences, Richmond, California, 94804, USA.
McCaman
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MT
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006801:Humans; D009930:Organic Chemicals; D012313:RNA / Q000032:analysis; D014771:Virion / Q000737:chemistry*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D012313:RNA