中国畜牧兽医2016，Vol.43Issue(9)：2230-2239,10.DOI:10.16431/j.cnki.1671-7236.2016.09.003
鉴别猪瘟野毒与疫苗毒的单一与二重 RT-PCR检测方法的建立及应用
Establishment and Application of Single and Duplex RT-PCR Method for Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus
摘要
Abstract
For the distinguishment of wild-type and vaccine viruses of classical swine fever (CSF), a rapid and simple method was established,which laid the foundation for the purification of classi-cal swine fever virus (CSFV)in large-scale farms.It was found that T-rich insertion sequence in-dependently that 3′-NTR in Lapinized vaccine strain according to the genome sequence compara-tive analysis of CSF wild virus,vaccine virus and near origin virus published in GenBank,on the basis of this,two pairs of specific primers were designed in the upper and lower ends of the inser-tion sequence and duplex RT-PCR primers were designed.The anneal temperature of polymerese chain reaction (PCR)was optimized,then the two single and duplex RT-PCR method for the dif-ferentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specifici-ty results showed that the minimum amounts of nucleic acid by the single RT-PCR were 2.2 pg (one pair of primers in single RT-PCR)and 1.7 pg (another pair of primers in single RT-PCR), respectively,and that of the duplex RT-PCR were 8.2 pg (wild-type virus of CSF)and 6.7 pg (vaccine virus of CSF),respectively,and no amplification of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods.Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rates of the mixed infection by wild-type and vaccine viruses of CSF in breeding sows,fattening pigs,nursery pigs and suckling piglets were 6.3%,7.4%,8.3%,8.6%,respectively.The results indicated that the single and duplex RT-PCR methods had high specificity,sensitivity,and good repeatability,and it had an important ref-erence value for the purification of CSF in large-scale farms.关键词
猪瘟病毒/野毒/疫苗毒/单一 RT-PCR/二重 RT-PCR/鉴别诊断/净化应用Key words
classsical swine fever virus/wild-type/vaccine viruses/single RT-PCR/duplex RT-PCR/differential diagnosis/purification and application分类
农业科技引用本文复制引用
马萍,李达,汤德元,张元鑫,张华,曾智勇,刘霞,韦冠东..鉴别猪瘟野毒与疫苗毒的单一与二重 RT-PCR检测方法的建立及应用[J].中国畜牧兽医,2016,43(9):2230-2239,10.基金项目
贵州省2014年农业攻关项目(黔科合 NY 字[2014]3055号) （黔科合 NY 字[2014]3055号）
贵州省科学技术基金项目(黔科合 J 字[2013]2110号) （黔科合 J 字[2013]2110号）
贵州大学研究生创新基金(农研[2015]030号) （农研[2015]030号）
