基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立

Establishment and Application of Fluorescence Quantitative PCR Detection Method based on Lumpy Skin Disease Virus ORF61 Gene

基于国内流行的牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)的基因组序列,建立了靶向ORF61基因的MGB探针荧光定量PCR检测方法。本研究基于LSDV基因序列设计出带有MGB探针的特异性荧光定量PCR引物,并进行特异性和灵敏性筛选验证。最终,筛选得到靶向于LSDV ORF61基因的一对qPCR引物;利用pCAGGS-ORF61真核表达质粒,获得标准曲线y=-3.287 5x+47.87,线性相关系数R^(2)=0.998 6,扩增效率达101%;荧光定量PCR特异性、重复性和灵敏性试验结果表明,该引物的特异性高、重复性良好和灵敏度高等优点;该引物的最低检测限为6.71拷贝·μL^(-1),各批次内与批次间重复性结果的变异系数均小于2%。以上结果表明,作者建立特异性LSDV的荧光定量PCR检测方法,为LSD预防和控制提供有效的检测手段。

Based on the genomic sequence of a prevalent Lumpy skin disease virus(LSDV)in China,we established an MGB probe fluorescence quantitative PCR method for detecting the ORF61 gene.In this study,a specific fluorescent quantitative PCR primer with an MGB probe was designed based on the LSDV gene sequence,and the specificity and sensitivity of this primer pair were screened and verified.Finally,we screened a pair of qPCR primers targeting the LSDV ORF61 gene.Using p CAGGS-ORF61,a eukaryotic expression plasmid,the standard curve y=-3.2875x+47.87 was obtained,the linear correlation coefficient was R^(2)=0.9986,and the amplification efficiency reached 101%.The results of the specificity test,repeatability test,and sensitivity test showed that this fluorescent quantitative PCR primer pair had the advantages of high specificity,good repeatability,and high sensitivity.The minimum detection limit of this primer pair was 6.71 copies·μL^(-1),and the coefficient of variation of intra-batch and inter-batch repeatability results was less than 2%.The above results indicate that we have established a fluorescence quantitative PCR method for the specific detection of LSDV,providing an effective detection method for preventing and controlling LSD.