摘要
从3个临床发病猪场采集三带喙库蚊提取RNA样本,采用针对猪繁殖与呼吸综合征病毒(PRRSV)NSP2基因设计的引物进行RT-PCR检测,并进行克隆、测序和序列分析;同时取三带喙库蚊匀浆上清接种Marc-145细胞进行PRRSV的分离与鉴定。结果表明:从三带喙库蚊提取的3份RNA样品均扩增出大小约为1 060 bp的特异性DNA条带,其序列与从相应猪场病猪分离得到的猪源PRRSV毒株高度同源,与国内参考株CH-1a株相应序列比较均缺失90个碱基。将3份三带喙库蚊匀浆上清接种Marc-145细胞后,均可观察到明显的细胞病变;以间接免疫荧光试验进行检测,可观察到培养细胞内的特异性荧光;采用RT-PCR反应可检出培养细胞内的PRRSV核酸。上述试验证实,三带喙库蚊可携带PRRSV,可能是引起PRRS传播与流行的重要途径之一。
The NSP2 gene fragments of porcine reproductive and respiratory syndrome virus （PRRSV） were amplified by RT PCR from the extracted RNA samples of Culex tritaeniorhynchus collected from 3 infected pig farms, and then were cloned into pMD-18T vector for sequence analysis. And the homogenate supernatant of Culex tritaeniorhynchus samples were inoculated to the Marc-145 cells for isolation and identification of PRRSV. The results showed that the specific DNA fragments of about 1 060 bp were amplified from the RNA samples from Culex tritaenio- rhynchus, and the sequences of these fragments shared high homology with the NSP2 gene of PRRSV isolates from infected pigs in the same farm. And there are 90 nucleotides deletion in NSP2 gene compared with CH la strain of PRRSV. The obvious cytopathic effect were appeared in Marc-145 cells inoculated with the samples of Culex tritaeniorhynchus, and the specific fluo- rescence were observed in the infected cells by indirect immunofluorescent assay, and the PRRSV-N gene fragments were amplified by RT-PCR from infected cells. These results indicated there are PRRSV in Culex tritaeniorhynchus, and it could be an important way of prevalence and transmission of PRRS.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2009年第2期280-284,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
贵州省"十一.五"农业科技攻关重大项目子课题[黔科合NZ字(2005)3002]
贵州省农业厅兽医科技计划项目[畜牧兽医专项20070717]
贵阳市农业局科技计划项目[兽医专项2007-08]
