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

Si, Wei; Zhou, Shun; Wang, Zhao; Cui, Shang-jin

With the recent availability of genomic sequences, molecular diagnostic methods for detection of viruses have significantly improved [15] . Complementary DNA and RNA probes have been used to detect RNA and mRNA of the CDV genome with improved specificity and sensitivity [6, 7] . Primers P1 and P4 (specific for CDV and conserved among CDV species), primer P2 (specific for CDV wild-type strain), and primer P3, P5 (specific for CDV vaccine strain), were selected from the well-conserved regions of the gene encoding matrix protein. A fragment of 600 bp was consistently amplified by RT-PCR with P1/P4 for either CDV vaccine strain or wild-type strain. The nested PCR with P2/P4 generated a 247 bp fragment only for the wild-type strain, while P3/P4, P5/ P6 generated a same 177 bp fragment only for the vaccine strain, and both fragments could be amplified from the mixture of CDV vaccine and wild-type strains. No amplification was obtained for NDV and other common canine viruses, such as CCV, CPV, CAV, RV, and uninfected cells control, indicating the high specificity of the method. The method was sensitive, in that it could detect as little as 0.1 TCID 50 of the virus. The selected two samples were classified into a branch which belongs to the wild-type strain, but constituting a genotype different from that of CDV vaccine strains, as revealed by phylogenetic analysis based on the sequences of the H gene region, which is believed to be the most reliable classification and genetic typing. RT-nPCR was used to detect the 30 field samples in Heilongjiang and Jilin provinces; 20 of the field samples were CDV-positive, among which 15 were wild-type strain, and 5 were vaccine strain.