A novel set of real-time PCRs for rapid differentiation between human cytomegalovirus wild-type and highly passaged laboratory strains

Abstract

Restricting amplification of human cytomegalovirus (HCMV) DNA to wild-type (WT) HCMV is useful to exclude PCR contaminations by laboratory strains from viral cultures. A set of UL141-specific TaqMan PCRs was developed to amplify (1) WT HCMV and laboratory strain Towne(long), but not strain AD169, (2) only WT HCMV. The performance was compared to a PCR targeting the conserved sequence of HCMV glycoprotein B using 46 serum and urine samples from blood donors with primary CMV infection. Amplification was restricted to the targeted strains with the exception of Towne(long) being amplified also by PCR (2), but at a distinctly lower efficiency than WT HCMV. The coefficient of regression for linear dilutions of two clinical samples with a high concentration of HCMV DNA was 0.999 and 0.997, respectively. The correlation between both WT PCRs and the generic HCMV PCR was good, with coefficients of regression of 0.891 and 0.871 for PCR (1) and (2), respectively. The limit of detection was calculated to be 1.5 genome equivalents per PCR. The set of HCMV TaqMan PCRs enables rapid differentiation between WT and laboratory strains, which can be especially useful as even virus lysate can contaminate sensitive PCRs without prior DNA isolation. A standardized WT HCMV control would be useful to evaluate WT-specific PCR methods.