猪细环病毒2型实时荧光定量PCR检测方法的建立  

Development of a real-time PCR assay for detection of Torque teno sus virus type 2

作　　者：单晶晶[1] 王俊[1] 孟凡伟[1] 于红欣[1] 周双海[1] 

机构地区：[1]北京农学院动物科学技术学院,北京102206

出　　处：《中国兽医科学》2014年第2期146-151,共6页Chinese Veterinary Science

基　　金：北京市属高等学校高层次人才引进与培养计划项目(CIT&TCD201304092)

摘　　要：根据猪细环病毒2型(TTSuV2)基因非编码区保守序列设计了1对特异性引物,构建含有TTSuV2基因片段的重组质粒,以系列稀释后的重组质粒作为模板来建立定量检测TTSuV2的SYBR GreenⅠreal-time PCR方法。结果显示,该方法的标准曲线的决定系数达0.999,显示出优良的线性关系;最低可检测50copies/μL的核酸模板,重复性试验的变异系数小于3%;用该方法对TTSuV1、猪圆环病毒、猪细小病毒、伪狂犬病病毒、猪瘟病毒及猪繁殖与呼吸综合征病毒等的检测结果均为阴性;对临床样品的检出率高于常规PCR方法的检出率。结果表明,建立的real-time PCR检测方法特异性强、灵敏度高、重复性好,可用于TTSuV2的检测与定量分析。The aim of this study was to establish a SYBR Green Ⅰ real-time PCR assay for quantitative detection of Torque teno sus virus type 2（TTSuV2）. A pair of primers specific to the conserved untransla-ted region of TTSuV2 gene was designed to construct a recombinant plasmid,and the positive recombinant plasmid was diluted serially as templates to develop a real-time PCR assay for quantitative detection of TTSuV2 based on SYBR Green Ⅰ. The coefficient for the developed standard curve was 0. 999,which indi-cated excellent linear relationship. The assay had a detection limit of 50 copies/μL of initial templates,and its coefficient of variation was less than 3% in the reproducible assays. No amplification was detected by this method from unrelated swine virus samples,including TTSuV1, porcine circovirus, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus. The detection rate of several clinical samples with this assay was higher than that with conventional PCR. The results indicated that this SYBR Green Ⅰ real-time PCR assay was of high specificity, sensitivity and reproducibility,and could be used for the detection and quantitative analysis of TTSuV2.

关 键 词：猪细环病毒2型 real—time PCR SYBR GreenⅠ 定量检测 

分 类 号：S852.659.2[农业科学—基础兽医学]