牛结节性皮肤病病毒荧光定量PCR方法的建立与应用

Development and Application of a Real-Time PCR Method for Detection of Lumpy Skin Disease Virus

为了建立牛结节性皮肤病病毒(LSDV)的快速诊断方法,本试验依据LSDV GCPR基因的保守序列区域设计了荧光定量PCR的引物和探针,并建立了快速诊断LSDV的荧光定量PCR方法。结果表明,建立的LSDV荧光定量PCR方法在3.67×10^(1)~3.67×10^(7) copies/μL拷贝标准品间具有良好的线性关系,线性相关系数达0.9905;该方法具有良好的敏感性,其检测病毒系列含量下限为3.67×10 copies/μL;该方法特异性良好,与山羊痘病毒、牛传染性鼻气管炎病毒、牛副流感病毒3型、牛病毒性腹泻病毒、牛呼吸道合胞体病毒等病毒不存在交叉反应;该方法重复性较好,批内和批间的变异系数均介于0.133%~1.81%。该方法,临床样品的检测符合率达90%。本试验所建立的LSDV荧光定量PCR方法为临床诊断牛结节性皮肤病提供了一种快速、灵敏的检测方法,为LSDV流行病学调查和监测提供了技术支持。

To develop a rapid diagnostic method for lumpy skin disease virus(LSDV),a real-time PCR method was developed using the primers and probe designed on the conserved sequence of GCPR gene.The standard curve of the real-time PCR assay had good linear relationships in the range of 3.67×10^(1)-3.67×10^(7)copies/μL of template with the correlation coefficient to 0.9905.The assay had good sensitivity of 3.67×10 copies/μL.There was no cross-reaction with goat pox virus,bovine viral diarrhea virus,infectious rhinotracheitis virus,bovine parainfluenza virus type 3 and bovine respiratory syncytial virus.The assay was reproducible 0.133%to 1.81%of the coefficients of valuation within a batch and between batches.The agreement rate of this real-time PCR was 90%in testing clinical samples.In conclusion,the real-time PCR assay developed here could be used as a diagnostic tool for LSDV,thus provided a technical support for the epidemiological investigation of LSDV.