中国兽医科学2011，Vol.41Issue(1)：60-64,5.
猪血凝性脑脊髓炎病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立
Development of a SYBR Green Ⅰ real-time PCR assay for detection of porcine hemagglutinating encephalomyelitis virus
摘要
Abstract
With a pair of primers designed according to the conserved sequence of S gene of hemagglutinating encephalomyelitis virus(HEV) in GenBank, the S gene was amplified by PCR and the recombinant plasmid containing the gene was constructed as a standard control. Then,a real-time PCR assay based on SYBR Green Ⅰ for HEV detection was developed. In result,the developed assay had a good linear relationship between initial templates and Ct values, and the correlation coefficient of the standard curve was 0. 994. The assay had high sensitivity and could detect 1.6 × 104 copies/μL. The specificity of the assay was high, so there was no cross reaction between HEV and other pathogens including bovine coronavirus,pseudorabies virus,porcine reproductive and respiratory syndrome virus. In addition, the assay had good reproducibility and there was a coefficient of variations less than 2％ for both intra- and inter-assay. 16 out of 20 suspicious positive samples detected by the real-time PCR were positive,while 8 out of the 20 suspicious positive samples detected by the conventional PCR were positive. These results showed that the developed SYBR Green Ⅰ real-time PCR assay had the advantages of rapid detection, specificity, sensitivity and good reproducibility,and it could be applied for the clinical diagnosis and quantification analysis of HEV.关键词
猪血凝性脑脊髓炎病毒/S蛋白基因/荧光定量聚合酶链反应分类
农业科技引用本文复制引用
臧德跃,高丰,王栋,李明谦,赵魁,张冰冰,陆慧君,贺文琦,陈克研,岳占碰..猪血凝性脑脊髓炎病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立[J].中国兽医科学,2011,41(1):60-64,5.基金项目
国家自然科学基金资助项目(30871849,31072134) （30871849,31072134）
吉林省科技发展国际合作项目(20080722) （20080722）
吉林省青年科研基金项目(20090154) （20090154）
