犬瘟热病毒弱毒和野毒的全基因组扩增及序列测定

Complete genome amplifications and sequencing of canine distemper virus vaccine strains and wild-type strains

分析比对了GenBank中已公布的23株犬瘟热病毒全基因组序列,针对保守区设计了11对特异性通用引物,用于扩增犬瘟热病毒野毒株和疫苗株全基因组。通过优化11对引物的工作浓度及退火温度等条件,对保存的3株疫苗毒CDV3株、OR12株、CDV/R-20/8株和2株野毒HBF-1株和SD(14)07株进行了全基因组扩增。取5μL扩增产物进行1%琼脂糖凝胶电泳,结果显示11对通用引物对以上5株犬瘟热病毒扩增后,均获得11个特异性目的片段,条带大小与预期相符。选取疫苗毒OR12株和野毒HBF-1株的进行全基因组序列测定,与GenBank中已公布的序列进行比对,同源性均大于99.0%,证实这11对特异性通用引物能够实现对犬瘟热病毒野毒和弱毒全基因组的准确扩增,并获得准确的全基因组序列。

This study compared and analyzed the complete genome sequences of 23 canine distemper viruse strains published in GenBank,and designed 11 pairs of specific universal primers for the conserved region to amplify the genome-wide sequence of canine distemper virus wild-type strains and vaccine strains.By optimizing the 11 pairs of primers working concentration and annealing temperature and other conditions,full-length genome sequence was amplified for three vaccine strains CDV3,OR12,CDV/R-20/8 and two wild-type HBF-1 and SD(14)07 preserved in our laboratory.5μL of the amplified product was subjected to 1%agarose gel electrophoresis,the results showed that the above five canine distemper viruses were amplified by using 11 pairs of universal primers,and 11 specific bands with the expected size were obtained.The whole genome sequence of the vaccine strain OR12 and the wild-type HBF-1 strain was selected and compared with the published sequences in GenBank,the homology was greater than 99.0%,It was confirmed that 11 pairs of specific primers designed by us can accurately amplify the full-length sequences of canine distemper virus wild strains and vaccine strains,and obtain accurate full-length sequences.