鉴别猪瘟病毒野毒株和兔化弱毒疫苗株的复合实时荧光定量RTPCR方法的建立与评价-预防兽医学专业论文.docxVIP

AbstractClassical Abstract Classical swine fever(CSF)，caused by Classical swine fever virus(CSFV)，is a highly contagious disease ofpigs，and one ofOIE Listed diseases．In recant years，CSF has changed a lot in clinical manifestations under the pressure of mass vaccination in China,resulting in atypical chronic forms quite different from acute CSF，and causing great economic losses in pork industry． CSFv．together with Bovine viral diarrhea virus l(BVDV-1)，BVDV-2，Border disease virus(BDV) and Pestivirus of giraffe，belongs to the genus Pestivirus within the family Flaviviridae．Pigs can be infected with almost any pestivirus species．It is rather difficult to differentiate CSFV from other pestiviruses by conventional serological tests due to cross—reactivity．On the other hand, extensive vaccination with C-strain vaccine makes it di硒cult to differentiate the animals infected with wild-type viruses from C—strain vaccine vaccinated ones using conventional assays． This study is aimed to develop a multiplex real—time RT-PCR for the quantitative and differential detection ofwild-type viruses and C-strain vaccine ofCSFV．All genomic sequences of pestiviruses available in GenBank including 34 strains CSFV genomic sequences were aligned． CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C—strain vaccine were designed in the 5-UTR ofthe viral genome ofCSFV． The two TaqMan probes specifically hybridize wild—type CSFV of different subgroups or C—strain vaccine virus，respectively，in the multiplex real-time RT-PCR,with no cross-reaction to other viruses of porcine origin．Completely correct differentiation of wild-type CSFV of different subgroups(1．1，2．1，2．2 and 2．3 subgroups)from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV．The坤was all excellent linear relationship from 10。to 10‘copies／pL when the positive standards ofwild-type and C·strain-type viruses were detected si