Development of an internally controlled, homogeneous polymerase chain reaction assay for the simultaneous detection and discrimination of herpes simplex virus types 1 and 2 and varicella-zoster virus

Homogeneous polymerase chain reaction (PCR) technology is being used increasingly in the diagnosis of infectious disease. The sensitivity and specificity of PCR is being coupled to the ease-of-use and multiplexing capacity of homogeneous methodologies to provide rapid and accurate differential diagnoses. This technology is applicable to the diagnosis of infections with the human herpes viruses, herpes simplex virus 1 (HSV 1), HSV 2 and varicella-zoster virus (VZV). Our aim was to develop and evaluate a homogeneous PCR assay which combines the following features: the assay can detect and distinguish HSV types 1 and 2 and VZV, can be performed on untreated clinical samples, contains internal control reagents to monitor for inhibitors in the sample and allows automatic assignment of viral genotypes. Primers and probes specific for HSV and VZV genes were combined and optimized in a multiplex PCR. An internal control was designed which allowed use of the VZV primers and a human factor V gene DNA template. The assay was evaluated on an initial cohort of 66 clinical swab samples, with results determined by visual inspection of melt curves. Parameters obtained from this study were used to assign genotypes automatically to a second group of 85 clinical swab samples. Optimization of reagents produced melt curve peaks of sufficient height and symmetry for automatic genotype assignment. In the initial cohort of 66 samples, 63 returned concordant results, one sample produced an aberrant peak due to sequence variation and the remaining two samples were positive on re-test. Automatic genotype assignment of the second group of 85 samples resulted in correct identification of 79 samples, with two further aberrant peaks, and two samples positive on retest. The development of this assay should facilitate the rapid detection of herpes viruses from clinical swab samples.