Establishment And Preliminary Application Of A Multiplex RT-nPCR For The Detection And Differentiation Of Wild-type Strains From Vaccine Strains Of Canine Distemper Virus

Canine distemper (CD) is a highly contagious and fatal disease of dogs or other furry animals by canine distemper virus (CDV), which is a single-stranded negative RNA virus belong to the Morbillivirus genus within the paramyxoviridae family. Other members of the genus are measles virus (MV) and riderpest virus (RPV). The genome of CDV is approximately 15690 nt in length, which contain some genes such as N, P, M, F, H, L. There only one serotype has been characterized.A multiplex RT-nPCR method was developed for the detection and differentiation of wild-type strains from vaccine strains of CDV. A pair of primers P1 and P4 specific for CDV corresponding to the highly conserved regions of CDV genome was used as a common primer pair in the first round PCR of the nested 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 of the nested PCR. A fragment of 177 bp was amplified from vaccine strain genome, and a fragment of 247 bp from wild-type strain genome in the RT-nPCR, and two fragments of 247 bp and 177 bp were simultaneously amplified from mixed genomic samples of vaccine strain and wild-type strain. No amplification was achieved for uninfected cells, or cells infected with newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), canine adenovirus(CAV). The sensitivity of RT-nPCR could reach 1×10^-1 TCID₅₀. The detection could be finished within six hours. The RT-nPCR method was used to detect the 30 field samples suspected of CD from Heilongjiang Province and Jilin Province. As a result, 20 samples were fould to be CDV, 15 field CDV samples were found to be wild-type strain, and 5 samples to be vaccine strain.The phylogenetic tree was obtained by selecting two samples (HLJ-1,HLJ-2) which were detected to be wild-type strain by RT-nPCR, and comparing the characteristic properties of the 25 strains which were representative for CDV strains from GenBank for H gene, then, as a result of this study, according to the phylogenetic tree of the H gene, The result of the RT-nPCR was right and the CDV strains could be divided into two genotypes which were about the wild-type strain and the vaccine strain, and there were five genotypes in the wild-type strain which were about the Asia-1 strain, Asia-2 strain, Europe strain, USA strain, Arctic strain, three strains (giant panda, TN, Guizhou) which were from China were in the lineage of the Asia-1, another strain (Liu) which was also from China was in the lineage of the Arctic, HLJ-1 and HLJ-2 were in the lineage of the Asia-2 strain.The RT-nPCR method could be used to effectively detect and differentiate wild-type CDV infected dogs from dogs which were vaccinated with CDV vaccine, thus could be used on the clinical detection and the epidemiologic monitoring.