A multiplex PCR assay for the simultaneous detection of human herpesvirus 6 and human herpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products

Abstract

A multiplex polymerase chain reaction (PCR) method was developed for the simultaneous detection of human herpesviruses 6 and 7 (HHV-6; HHV-7) in clinical samples, using primers which amplify a section of the HHV-6 U67 gene and the HHV-7 homologue of the HHV-6 U42 gene. Comparison of the multiplex assay with the respective single PCR assays, using cloned HHV-6 and HHV-7 sequences as targets for amplification, showed equivalent sensitivity and specificity for the assays. To demonstrate the use of multiplex PCR for the analysis of clinical samples, serum and saliva from infants were analysed using this technique. The results showed that a clear distinction can be made between the amplicons of HHV-6 and HHV-7, without loss of sensitivity or specificity. There was complete concordance between the respective single PCR assays, and the multiplex PCR. HHV-6 amplicons derived from the multiplex PCR analysis were typed by differential AvaII restriction endonuclease digestion, in which HHV-6 variant A amplicons are cleaved but those of variant B remain undigested. These results were compared to HHV-6 variant typing by an established method, the results of which showed complete concordance between assays. It is proposed that this multiplex assay, where HHV-6 positive samples may be typed directly from the reaction products, is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of HHV-6 and HHV-7.