猪源牛病毒性腹泻病毒套式PCR检测方法的建立

Establishment of Nested-PCR for detecting bovine viral diarrhea viruses from pig

根据GenBank中BVDV-1、BVDV-2、CSFV及新出现瘟病毒的基因序列,设计2对特异引物,建立了能鉴别诊断BVDV与CSFV的Nested-PCR检测方法;确定其方法的特异性、敏感性、稳定性。建立的Nested-PCR能从BVDV标准毒株、猪源BVDV毒株中扩增198bp特异性片段,对猪瘟病毒、猪繁殖与呼吸综合征病毒检测结果为阴性。结果表明,该方法具有较好特异性;其敏感性可检测到0.195fg RNA模板量;经重复性试验表明该方法具有良好稳定性。初步应用检测表明,猪BVDV阳性率较高,达36.0%;牛血清及其制品、猪瘟活疫苗BVDV污染严重。本试验建立的Nested-PCR具有敏感、特异和稳定等特点,可用于临床诊断和流行病学调查。

The study aimed at establishing Nested-PCR to differentiate pig bovine viral diarrhea viruse（BVDV） and classical swine fever virus（CSFV）,for clinical diagnosis,epidemiological survey and quality control of classical swine fever live vaccine.Two pairs of primers were designed according to the genomic sequences of BVDV-1,BVDV-2,CSFV and a new member of the pestivirus genus,and a Nested-PCR were developed for the differentiation of pig BVDVs and CSFV.This PCR assay could respectively amplify a 198 bp fragment from BVDV NADL,pig BVDV,but not from CSFV,porcine reproductive and respiratory syndrome virus （PRRSV）.Sensitivity was determined as 0.195 fg RNA.And in the field trail of 100 pig specimen,36% of them were positive.The results showed that the Nested-PCR with high sensitivity and specificity could provide a new and alternative tool for the detection of pig BVDVs and CSFV.