中国兽医科学2017，Vol.47Issue(6)：687-693,7.DOI:10.16656/j.issn.1673-4696.2017.06.003
兔出血症病毒可视化RT-LAMP检测方法的建立与应用
Development and application of RT-LAMP for visual detection of rabbit hemorrhagic disease virus
摘要
Abstract
To establish a visible RT-LAMP for detection of rabbit hemorrhagic disease virus,according to the sequence of major structural protein VP60 gene of RHDV in GenBank,a set of four specific primers was designed and synthesized.Using recombinant pMD19-T-VP60 plasmid as a template,the LAMP for detection of RHDV was developed by optimizing its reaction system and conditions.lts detection limit for RHDV was determined by detecting the fold-diluted pMD19-T-VP60 plasmid.The optimized reac tion temperature and time of RHDV-LAMP were at 62℃ and 40 min,respectively.The detection limit of the LAMP assay was 50 copies/μ L,while the detection limit of the conventional PCR was 5 × 103copies/μ L,suggesting that the RT-LAMPmethod was more sensitive than the traditional RT-PCR.The LAMP amplification products was visually inspected after addition of ethidium bromide:positive samples turned pink,while negative samples and no template control reactions remained orange.Moreover,no amplification was found with the samples of Pasteurella,Escherichia coli,Salmonella,Clostridiun welchii,Streptococcus agalactiae and Klebsiella pneumoniae.The above-mentioned results showed that the developed RT-LAMP in this study was more convenient,rapid,highly sensitive and specific than that of the traditional RT-PCR,and it did not require expensive equipment,which was more suitable for field testing.关键词
兔出血症病毒/RT-LAMP/RT-PCR/VP60基因Key words
rabbit hemorrhagic disease virus (RHDV)/loop-mediated isothermal amplification (LAMP)/RT-PCR/VP60 gene分类
农业科技引用本文复制引用
刘星星,耿毅,孙娜,陈勤,杨泽晓,李亚军,牟维豪..兔出血症病毒可视化RT-LAMP检测方法的建立与应用[J].中国兽医科学,2017,47(6):687-693,7.基金项目
四川省科技支撑计划项目(No.2016NZ0002) （No.2016NZ0002）
