猪瘟病毒NASBA-ELISA检测方法的建立与应用

为建立猪瘟病毒NASBA-ELISA检测方法,以猪瘟病毒(CSFV)E2基因为研究对象,利用Prim-er 5.0和Blastn生物软件,设计并合成了特异的核酸序列依赖扩增(NASBA)引物和探针。经条件优化,确定的最佳扩增反应条件为42℃,反应1.5h。临床检测结果显示,用该方法可特异地扩增CSFV E2基因约155bp的目的片段,而对TGEV、PRRSV、PCV2、PPV、PEDV、APP和PK-15正常细胞等核酸的检测均为阴性,可最低检出1×100～1×101 copies/μL,其灵敏度比RT-PCR略高。应用该方法对采自重庆部分地区猪场的125份样品进行了检测;结果,猪瘟病毒阳性检出率为5.6%,NASBA-ELISA法与RT-PCR法的检出符合率为94.4%。结果表明,NASBA-ELISA方法可应用于CSFV的快速鉴别检测。

Establishment and application of an NASBA-ELISA for detection of classical swine fever virus

In order to establish a nucleic acid sequence-based amplification(NASBA) method for the detection of classical swine fever virus(CSFV),specific NASBA primers and probe of CSFV E2 gene were designed using Primer 5.0 and Blastn.Based on solution hybridization and enzyme-linked technology,the NASBA-ELISA method for the detection of CSFV were developed successfully,and the optimized amplification condition of nucleic acid isolation is 42 ℃ and 1.5 h.The result of clinical detection indicated that specific fragments of 155 bp were obtained from the CSFV positive samples by NASBA,other than in TGEV,PRRSV,PCV2,PPV,PEDV,APP and PK-15 cell samples.The NASBA assay had a detection limit equivalent from 1 to 10 CSFV copies/μL.The sensibility of NASBA-ELISA was similar to that of the RT-PCR.A total of 125 clinical samples of pigs were analyzed for CSFV with a positive rate of 5.6%,which showed that this assay had a coincidence rate of 94.4% with NASBA-ELISA and RT-PCR.In conclusion,this NASBA-ELISA method was simple to perform and can be applied to the rapid identification and detection of CSFV.