猪圆环病毒2型LAMP检测方法的建立与应用  

Development and application of LAMP method for detection of porcine circovirus type 2

作　　者：杨泽晓[1,2] 侯义宏 曾辉[1] 姚学萍[1] 王印[1] 刘红亮[1] 刘波[1] 邬旭龙[1] 彭彬[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014 [3]常德出入境检验检疫局,湖南常德415100

出　　处：《中国兽医科学》2014年第2期152-158,共7页Chinese Veterinary Science

基　　金："十二五"国家科技支撑计划项目(2013BAD12B04);水禽疫病防控四川省青年科技创新研究团队项目(2013TD0015);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848)

摘　　要：为建立快速检测猪圆环病毒2型(PCV2)的LAMP方法,根据GenBank发表的PCV2ORF2基因组保守序列,设计合成4条特异性LAMP引物,通过PCV2ORF2的制备、反应条件的优化、敏感性试验和特异性试验,初步建立了PCV2LAMP检测方法,并对60份临床检样和6株PCV2进行了检测应用。结果显示,建立的PCV2LAMP检测方法具有良好的特异性和敏感性,在64℃45min条件下可扩增出大于134bp的特异性梯状DNA条带,检测限度可达10copies/μL;用它对PCV1、PRV、PPV、CSFV和PRRSV的核酸进行扩增,但结果均为阴性。用建立的LAMP方法进行的样品检测结果显示,6株受检PCV2毒株的检出率为100%,60份临床检样PCV2的阳性率为20%(12/60),与PCR方法平行检测的结果相一致。To develop a loop-mediated isothermal amplification（LAMP） method for quick detection of porcine circovirus type 2（PCV2） ,4 specific LAMP primers were designed and synthesized according to the conserved sequences of ORF2 gene of PCV2 published in GenBank. The LAMP assay was established and evaluated by the preparation of target gene fragments, optimization of reaction conditions, sensitivity and specificity tests. Then 6 strains of PCV2 and 60 clinical samples were detected using LAMP and PCR in parallel. In result,the LAMP method for the PCV2 detection performed at 64 ℃ for 45 rain showed a lad-der-like pattern of amplification bands more than 134 bp. The detection limitation reached 10 copies/μL, and the assay gave no amplification from PCV1, PRV, PPV,CSFV and PRRSV. PCV2 positive rate in the detected samples was 20 % and all the 6 strains of PCV2 were positive by the two methods. These results indicated the good correlation between PCV2 LAMP and PCR.

关 键 词：猪圆环病毒2型 LAMP 检测 

分 类 号：S852.659.2[农业科学—基础兽医学]