马立克氏病病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用

Establishment of a SYBR GreenⅠ-based fluorescent quantitative PCR assay for Marek’s disease virus

根据马立克氏病病毒PP38基因的一段特异性保守序列设计引物,将MDV的PP38基因构建到19T载体上,将获得的重组质粒作为标准阳性模板,建立了针对马立克氏病病毒的SYBR GreenⅠ实时荧光定量PCR检测方法,并对该方法的特异性、灵敏性和重复性进行了评价。结果显示,所建立的SYBR GreenⅠ荧光定量PCR方法的Ct值与标准品在6.53×10^(1)~6.53×10^(8)copies/μL范围内呈良好的线性关系,其线性相关系数R^(2)=0.998 8,标准曲线方程为Y=-3.271X+39.297,熔解曲线呈现单峰,无非特异性扩增。且检测J亚群禽白血病病毒(ALV-J)、新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、禽流感病毒(AIV)和鸡传染性贫血病毒(CIAV)时结果均呈阴性,具有良好的特异性。对20份临床组织样品检测结果进行分析发现,与普通PCR检测结果相比阳性率高出20%。本研究建立的检测MDV的SYBR GreenⅠ荧光定量PCR方法可用于马立克氏病病毒的快速诊断,对马立克氏病的监测具有重要意义。

Primers were designed according to a specific conservative sequence of PP38 gene of Marek’s disease virus.The PP38 gene of MDV was constructed on 19T vector.The recombinant plasmid was used as a standard positive template to establish a real-time fluorescence quantitative PCR method for detection of Marek’s disease virus SYBR GreenⅠ.The specificity,sensitivity and repeatability of the method were evaluated.The results showed that there was a good linear relationship between the Ctvalue of the established SYBR GreenⅠfluorescence quantitative PCR method and the standard sample in the range of 6.53×10^(1)—6.53×10^(8)copies/μL,the linear correlation coefficient was R^(2)=0.998 8,the standard curve equation was Y=-3.271X+39.297,and the melting curve showed a single peak without non-specific amplification.The detection results were negative for subgroup J avian leukemia virus (ALV-J),Newcastle disease virus (NDV),infectious bronchitis virus(IBV),avian influenza virus(AIV) and chicken infectious anemia virus(CIAV).After analyzing the results of 20 clinical tissue samples,it was found that the positive rate was 20%higher than that of ordinary PCR.The SYBR GreenⅠfluorescence quantitative PCR method established in this study for the detection of MDV can be used for the rapid diagnosis of Marek’s disease virus and is of great significance for the surveillance of Marek’s disease.