检测CSFV、JEV、PRRSV三种RNA病毒多重RT-PCR方法的建立  

Multiplex RT-PCR for detection of the three RNA viruses CSFV,JEV and PRRSV

作　　者：张雪寒[1] 何孔旺[1] 郭容利[1] 俞正玉[1] 倪艳秀[1] 

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014

出　　处：《中国预防兽医学报》2007年第5期385-388,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家863项目(编号:2001AA249012);国家攻关项目(编号:2004BA514A16-5)资助

摘　　要：猪瘟病毒(CSFV)、流行性乙型脑炎病毒(JEV)和猪繁殖与呼吸综合征病毒(PRRSV)是引起严重的种猪繁殖障碍的病原,而且经常混合感染,及时准确诊断是防治的前提。根据GenBank发表序列选取3对引物建立检测CSFV、JEV和PRRSV病毒的多重RT-PCR方法,扩增产物分别为508 bp、380 bp、263 bp。经与IDEXX商品化的检测CSW抗原试剂盒比较,二者的符合率为96.7%;扩增JEV和PRRSV PCR产物分别经EcoR V和Sau3A I酶切得到预期的片段。建立的多重RT-PCR检测JEV、PRRSV和CSFV敏感度分别为12.5个TCID_(50)、10个TCID_(50)和10^(-3)ng总RNA。结果表明该多重RT-PCR方法具有很好的特异性和敏感性,可用于临床三种病毒核酸的检测。Classical swine fever virus （CSFV）, Japanese encephalitis virus （JEV） and porcine reproductive and respiratory syndrome virus （PRRSV） are the major causes of some serious reproductive diseases and co-infection is common. Therefore rapid and reliable diagnosis is crucial for effective prevention and treatment. In this study, a multiplex PCR was developed using primers designed according to published CSFV, JEV and PRRSV sequences. PCR products of 508 bp, 380 bp and 263 bp were obtained for respective viruses. Restriction digestion of PCR products with EcoR Ⅴ and Sau3A Ⅰ produced expected target DNA fragments. Compared to CSFV antigen test kit, the multiplex RT-PCR showed 96.7 % coincidence with the commercial kit based on clinical test results. As low as 12.5 TCID50 JEV, 10 TCID50 PRRSV and 10^-3 ng total RNA including CSFV RNA could be detected by multiplex PCR. The result showed that the Multiplex RT-PCR is a rapid, sensitive and specific method for detecting virus from clinical samples.

关 键 词：猪瘟病毒 流行性乙型脑类病毒 猪繁殖与呼吸综合征病毒 多重RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学] S854.44[农业科学—兽医学]