猪瘟石门系强毒株SYBR Green-Ⅰ染料法实时荧光定量PCR方法的建立

Development of a Real-time Flourescent Quantitative PCR for Detection of Classical Swine Fever Virus Shimen Strain

为了能快速、准确地诊断猪瘟病毒(classical swine fever virus,CSFV)石门系强毒,根据登录在GenBank上23株Shimen毒株的全基因组序列,利用DNASIS软件进行序列分析,设计1对特异的荧光定量引物。建立猪瘟石门系强毒株SYBR Green-Ⅰ染料法实时荧光定量PCR快速检测方法。试验结果表明,本方法最低可检测到的Shimen病毒cDNA含量为100拷贝/μL,敏感度至少是普通PCR的100倍;猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪伪狂犬病病毒(PRV)、牛流行腹泻病毒Ⅰ型(BVDV-1)和猪瘟病毒C株均无特异性扩增,证明本方法具有很强的特异性;通过批内和批间重复试验,其变异系数<0.7%,结果显示本方法具有很好的重复性。本研究建立了猪瘟石门系强毒株SYBR Green-Ⅰ染料法实时荧光定量PCR快速检测方法,为猪瘟的预防与控制提供了有效的技术手段,并为猪瘟强毒试剂盒的研发奠定了基础。

In order to be able to fast and accurate diagnosis of classical swine fever virus strain Shimen based on the alignment of genomic sequences of 23 strains of Shimen available in GenBank,one pair of differential primers were designed by DNASIS software.SYBR Green-Ⅰ real-time PCR for detection of classical swine fever virus strain Shimen was established.The sensitivity was 100 copies/μL.The assay is highly specific and showed no cross reactivity against PRRSV,PCV2,PRV,BVDV-1 and C-strain.Inter-assay and intra-assay variables of CV were less than 0.7%.This method was highly sensitive specific and rapid.This assay could provide a powerful tool for quantification of Shimen-strain,and could be further developed into a commercial kit for rapid detection of Shimen-strain.