The Establishment of AS-PCR Method to Distinguish Canine Distemper Asia-I Type Wild Strains and Vaccine Strains |
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| Authors: | ZHANG Xueting CHEN Zhengrong ZHAO Wenwen HAN Li CHEN Jianguo |
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| Institution: | Huazhong Agricultural University, Wuhan 430070, China |
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| Abstract: | Canine distemper (CD) is a contagious disease, which can damage the immune system, respiratory system, digestive system, and even nervous system, leading to systemic pathological changes, and has a huge threat to pet dogs, fur animals, etc. At present, the commonly used colloidal gold test cannot effectively distinguish vaccine immunity from animal natural infection. To establish an efficient and accurate detection method for identifying wild strains and vaccine strains of canine distemper virus(CDV), the whole genome of six canine distemper wild strains isolated from dogs and three widely used CDV vaccine strains collected from Wuhan area were sequenced. After comparing and analyzing the amino acid and the base sequences, the H gene was determined as the target gene for AS-PCR primer design. By genotyping the H gene, it was found that the prevalent CDVs in Wuhan were all Asia-Ⅰ, while the vaccine strain Y2 was America-I, and the vaccine strains Y1 and Y3 were both America-Ⅱ. Comparing the CDV-H gene sequences of 179 Asia-Ⅰtypes (6 wild-type strain samples + 173 GenBank Asia-Ⅰtype stains) and 3 vaccine strains, using AS-PCR technology (3'mismatch) to design a pair of primers. It can effectively distinguish CDV Asia-I wild strain and vaccine strain. The upstream primer sequence is 5'-TTAATATAATAATGACAGTG-3', and the downstream primer sequence is 5'-CCTCAAGGGGCACA-3'. The results showed that the primer had a strong specificity and the wild strains could amplify an 894 bp fragment, while the vaccine strains could not. There are 9 more regular bases (amino acids) variation sites in H gene of wild strain, and the 277th amino acids of all vaccine strains are Asparagine. These mutations may lead to an increase in N-glycosylation sites, which will have an impact on the virulence of canine distemper virus vaccine strains. The AS-PCR method established in this study can effectively distinguish the canine distemper vaccine and Asia-Ⅰwild strains. |
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| Keywords: | canine distemper virus detection H gene AS-PCR |
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