A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

A multiplex reverse transcription-nested polymerase chain reaction(RT-nPCR) was developed for detection and differentiation of wild-type and vaccine strains of canine distemper virus(CDV).A pair of primers P1 and P4 corresponding to the highly conserved regions of CDV genome was used as common primers in the first round PCR.Primers P2 or P3 specific for CDV wild-type strain or vaccine strain respectively were used as the forward primer together with the common reverse primer P4 in the second round PCR.A PCR fragment of 177 bp from vaccine strain genome and a fragment of 247 bp from wild-type strain genome were amplified by the RT-nPCR.Two fragments of 247 bp and 177 bp were amplified from mixed genomic samples of vaccine strain and wild-type strain.No PCR products were detected for uninfected cells,or cells infected with newcastle disease virus(NDV),canine parvovirus(CPV),canine coronavirus(CCV),rabies virus(RV),canine adenovirus(CAV).The RT-nPCR method was successfully used to detect for suspected CDV infection in 20 clinical samples from Heilongjiang and Jilin Provinces,of which 15 samples were found infected by wild type CDV,and 5 by vaccine strains.This method was effective for detecting and differentiating wild-type CDV and vaccine strains in dogs,thus could be useful for the clinical detection and the epidemiological studies.It also can be used for monitoring the vaccinal immunity.