Quantitation of herpes simplex viral DNA in vero cells for evaluation of an antiviral agent using the polymerase chain reaction

A method for quantitation of the DNA of Herpes simplex virus type 2 (HSV-2) -infected Vero cells by the polymerase chain reaction (PCR was developed. T his method allowed recognition of several molecules of viral DNA among the total DNA extracted from cells. The method could be applied to a very large range (10(-0)-10(-7)) of initial amounts of template. Products of PCR were collected after each cycle for kinetic analysis. Poducts were subjected to electrophoresis and amplified bands were stained with ethidium bromide. Th e intensity of fluorescence of each band was measured with a charge-coupled device (CCD) image analyzer. The time course of increases in the relative yield of viral DNA was determined. Two-fold amplification of viral DNA occu rred each 6-h cycle from 7 h after infection. Using this method, the yields of viral DNA after treatment with the drug acyclovir (ACV) at 0.1 and 2 mu g/ml were about 1/10 and 1/80 of those from nontreated infected cells, res pectively. These results indicate that this method makes clear the inhibito ry effect of ACV on the synthesis of viral DNA. (C) 1998 Elsevier Science B .V. All rights reserved.