猪繁殖与呼吸综合征和猪乙型脑炎双重一步法RT-PCR诊断试剂盒的研制  

Development of dual one-step RT-PCR detection kit for PRRSV and JEV

作　　者：余波[1] 谭诗文[1] 冉懋韬[1] 徐景峨[1] 史开志[1] 杨丽娟[1] 

机构地区：[1]贵州省畜牧兽医研究所,贵州贵阳550005

出　　处：《广东农业科学》2013年第10期152-154,180,共4页Guangdong Agricultural Sciences

基　　金：贵州省科技厅农业攻关项目(黔科合NY字[2010]3085);贵州省畜禽健康养殖技术创新能力建设项目(黔科合院所创能[2010]4004);中央补助地方科技基础条件专项基金(黔科条中补地[2010]4001);贵州省农业科学院院专项(黔农科院院专项[2008]040)

摘　　要：根据GenBank中的猪繁殖与呼吸综合征病毒(PRRSV)ORF7 N基因、猪乙型脑炎病毒(JEV)NS1基因序列,设计了2对引物,通过反应条件的优化,成功研制了检测PRRSV和JEV的双重一步法RT-PCR诊断试剂盒,扩增产物分别为245、504 bp。敏感性、特异性结果显示,该RT-PCR诊断试剂盒对2种病毒的最低核酸检测量分别为PRRSV 0.2 ng/L、JEV 0.4 ng/L,而对猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型、猪细小病毒的扩增结果均为阴性。对52份疑似病猪样品的检测结果表明,该双重一步法RT-PCR诊断试剂盒检测结果与单一PCR检测结果完全符合。结果表明该双重一步法RT-PCR诊断试剂盒具有很好的特异性和敏感性,可用于临床猪繁殖与呼吸综合征和猪乙型脑炎的检测。to the gene sequences in GenBank of PRRSV ORF7 N gene and JEV NSI gene, two pairs of specific＇, primers were designed for amplil^ing the two specific fragments of PRRSV and JEV respectively. After optimization of annealing temperature and primers eoncentrations, dual one-step RT-PCR detection kit for PRRSV and JEV was development. Two specific bands of 245 bp （PRRSV）, 504 bp （JEV） were amplified. The sensitivity and specificity tests showed that the dual one-step RT-PCR was highly sensitive in PRRSV 0.2 ng/L, JEV 0.4 ng/L. No band was amplified from CSFV, PRV, PCV-2, PPV by the dual one-step RT-PCR. 52 clinical samples were deteclcd by the dual one-step RT-PCR detection kit for PRRSV and JEV. The results revealed that the dual one-step RT- PCR detection kit was sensitive, specific and reproducible and it could be used to detect rapidly PRRSV and JEV.

关 键 词：猪繁殖与呼吸综合征病毒 猪乙型脑炎病毒 双重一步法RT—PCR 

分 类 号：Q781[生物学—分子生物学]