Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent dye

A microplate assay for the rapid quantitation of adenovirus DNA has been de veloped 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 re lationship 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 pr esent 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 ar e lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 2 0-fold more sensitive. The dye binding assay also uses considerably less sa mple volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (t ypically 1.5- to 2-fold) than this spectroscopic method. The applications a nd limitations of this method in the analysis of adenovirus samples are dis cussed. (C) 1999 Academic Press.