Development of subfamily-based consensus PCR assays for the detection of human and animal herpesviruses

Abstract

Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10^-5 for alpha-HVs and 10^-3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10^-5, 10^-3, and 10^-2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.

Keywords: Consensus PCR; DNA polymerase gene; Glycoprotein B gene; Herpesvirus.

Fig. 1

Electrophoresis of PCR products of HV DNAs obtained by STC-PCR in a 1.5% agarose gel. Lane 1 and 18 contain a 100 bp DNA marker; Lane 2 = Bovine alphaherpesvirus 1; lane 3 = Chelonid alphaherpesvirus 5, lane 4 = Macropodid alphaherpesvirus 1; lane 5 = Macropodid alphaherpesvirus 2; lane 6 = Human alphaherpesvirus 1; lane 7 = Human alphaherpesvirus 2; lane 8 = Human alphaherpesvirus 3; lane 9 = Equid alphaherpesvirus 4; lane 10 = Meleagrid alphaherpesvirus 1; lane 11 = Gallid alphaherpesvirus 2; lane 12 = Felid alphaherpesvirus 1; lane 13 = Human betaherpesvirus 5; lane 14 = Human betaherpesvirus 6; lane 15 = Human betaherpesvirus 7; lane 16 = Human gammaherpesvirus 4; lane 17 = Human gammaherpesvirus 8