猪圆环病毒2型LAMP可视化快速检测方法的研究  

Visual rapid detection of porcine circovirus type 2 by a loop-mediated isothermal amplification assay

作　　者：胡瑞丽[1] 倪建平[2,3] 赵笑[4] 赵庆 史斌 赵桓震 赵斌安 赵凯[2,8] 

机构地区：[1]上海师范大学,上海200234 [2]上海市农业科学院生物技术研究所,上海201106 [3]上海佳牧生物制品有限公司,上海201106 [4]宁夏大学,银川750021 [5]上海斯高勒生物科技有限公司,上海200233 [6]上海珺珏生物科技有限公司,上海201501 [7]上海博满生物科技有限公司,上海201106 [8]上海市农业遗传育种重点实验室,上海201106

出　　处：《上海农业学报》2016年第1期23-28,共6页Acta Agriculturae Shanghai

基　　金：上海市闵行区重大产业化专项(2014MH076)

摘　　要：本研究旨在建立有效的针对猪圆环病毒2型(PCV2)的诊断技术及开发试剂盒。采用环介导等温扩增方法(LAMP)检测猪圆环病毒2型(PCV2),对PCV2的ORF2基因设计出3对特异性引物,扩增PCV2基因的最佳温度和时间优化到59℃孵育55 min。用该方法对临床样品进行检测,LAMP检测对PCV2的检测限为10拷贝/μL,而常规PCR检测法的检测限为1 000拷贝/μL,表明LAMP检测法灵敏度高。该检测法不会与猪圆环病毒Ⅰ型、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、传染性胃肠炎的猪病毒和轮状病毒发生交叉反应。用建立起的LAMP检测法对1 100个临床样品进行检测,950个样品被检测出为阳性样品。稳定性试验表明该试剂盒可以耐受至少40次的反复冻融。结果显示,该方法建立的猪圆环病毒2型诊断方法灵敏度高,特异性强,稳定性好。In order to establish effective diagnostic technique and diagnostic kit for porcine circovirus type 2（PCV2）,a loop-mediated isothermal amplification（LAMP）assay was used to detect PCV2,three pairs of specific primers were designed for PCV2＇ s 0RF2 gene,and the time and temperature conditions for amplification of PCV2 gene were optimized to be 55 min at 59 t.Testing clinical samples by this method showed that the detection limit for PCV2 was 10 copies/μL,whereas the conventional PCR detection limit was 1 000 copies/μL,indicating that the LAMP method was highly sensitive.The detection did not cross-react with PCVI,porcine reproductive and respiratory syndrome virus,porcine epidemic diarrhea virus,transmissible gastroenteritis＇ pig virus and rotavirus.When1 100 samples were tested by the established LAMP method,950 samples were detected to be positive.The stability test showed that this kit could endure at least 40-time reiterative freeze-thaw.The above results indicated that the established method and diagnostic kit had high sensitiveness,strong specificity and good stability.

关 键 词：猪圆环病毒2型 环介导等温扩增 检测 诊断试剂盒 

分 类 号：S852.65[农业科学—基础兽医学]