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

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.