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

After the application of the first-round PCR, primers P2 and P4, and P3, P4, P5 and P6 together, were used to amplify the vaccine and wild-type strains, respectively, at different anneal temperatures. According to the result, when the anneal temperature was from 49-54°C, there was only one specific band. Primers P2, P3, P4, P5 and P6 were used to perform RT-nPCR with different anneal temperatures. Only one specific band was observed at an anneal temperature from 49.5-54.5°C, with the most distinct band appearing at 51.5°C. Thus, 51.5°C was chosen for the RT-nPCR.