A TOUCHDOWN PCR FOR THE DIFFERENTIATION OF EQUINE HERPESVIRUS TYPE-1 (EHV-1) FIELD STRAINS FROM THE MODIFIED-LIVE VACCINE STRAIN RACH

More than 50 reference strains and field isolates of equine herpesviru s type 1 (EHV-1) were examined by a touchdown PCR. Primers for specifi c amplification of EHV-1 DNA were chosen from the terminal and interna l, repeat regions of the EHV-1 genome where the high-passaged live vac cine strain RacH displays symmetric 850 bp deletions. The positive str and and one negative strand primer were designed to encompass the dele tions present in RacH, and the second negative strand primer was desig ned to hybridize within these deletions. Discrimination between field isolates and the vaccine strain was achieved by the generation of ampl ification products of different size: In all EHV-1 reference strains a nd field isolates, a 495 bp DNA fragment was amplified specifically, w hereas a 310 bp fragment was amplified when DNA of the vaccine strain PacH was used as a template. PCR amplification was only obtained in th e presence of 8-10% dimethylsulfoxide and when the primer annealing te mperatures were decreased stepwise from 72 degrees C to 60 degrees C. Under these conditions as little as 100 fg template DNA, corresponding to about 100 genome equivalents, could be detected. The PCR assay all ows fast and sensitive discrimination of the modified live vaccine str ain RacH from field strains of EHV-1 since it is applicable to viral D NA extracted from organ samples and paraffin-embedded tissues. It may thus be helpful for examining the potential involvement of the RacH li ve vaccine strain in abortions of vaccinated mares.