摘要
为建立一种快速、特异、灵敏的检测猪瘟病毒(classical swine fever virus,CSFV)巢式RT-PCR(nested RTPCR)方法,本研究参照GenBank中公布的CSFVE2基因保守区域序列设计2对特异性引物,以CSFV总RNA为模板,优化反应条件,建立CSFV nested RT-PCR方法,对其进行特异性、敏感性和重复性试验;利用所建立的方法对35份临床疑似CSFV感染样品进行了应用检测,并对阳性PCR产物进行克隆测序鉴定。结果表明,本研究成功建立了CSFV nested RT-PCR检测方法,能够特异性地扩增CSFV,但对ST正常细胞对照和其他8种病原对照未扩增出任何条带;稳定性和重复性好;敏感性高,最低检测病毒含量为1TCID50;自35份疑似CSFV感染样品中检出19份阳性样品,PCR阳性产物克隆测序结果表明均为CSFV E2基因片段。本研究成功建立了CSFV nested RT-PCR检测方法,可用于CSFV的快速检测,为CSF的早期检测诊断提供了特异、灵敏的方法。
A nested RT-PCR for detection of classical swine fever virus（CSFV）was developed using the total RNA of CSFV as a reverse transcription template,and two pairs of specific primers were designed based on the conserved region of CSFV E2 gene.The specificity,sensitivity and repetition of CSFV nested RT-PCR were tested,and 35 samples taken from clinical suspicious CSFV infected pigs were tested by the established nested RT-PCR assay.The results indicated that the nested RT-PCR assay was successfully established.The sensitivity and specificity of the nested RT-PCR assay revealed that the minimum detectable virus content limit was 1TCID50,and no products were amplified from the nucleic acid of ST normal cell or other 8pathogenic viral or bacterial microorganisms.The repetition test indicated the nested RT-PCR assay showed a good repeatability.Meanwhile,19 positive samples were detected in 35 suspected samples,which was consistent with the results tested by sequencing.The study suggested that the established nested RTPCR assay was specific,sensitive and suitable for early detection of CSFV.
出处
《中国畜牧兽医》
CAS
北大核心
2016年第9期2285-2290,共6页
China Animal Husbandry & Veterinary Medicine
基金
河南省科技攻关项目"病死动物无害化处理技术及管理机制研究"(142102310224)
关键词
猪瘟病毒
巢式RT-PCR
检测
建立
应用
classical swine fever virus
nested RT-PCR
detection
establishment
application
