摘要
参考NCBI中已收录的鸭坦布苏病毒(DTMUV)E基因保守区域设计了6条引物,通过对反应体系中各组分和条件进行优化,建立了DTMUV环介导等温扩增(LAMP)检测体系,并应用荧光显色剂(SYBR GreenⅠ和钙黄绿素、锰离子)对扩增产物进行可视化判定。结果显示,以SYBR GreenⅠ为染料,显色敏感性为10copies/μL的病毒,比普通PCR高100倍;以钙黄绿素和锰离子组合作为显色剂,其显色极限为1 000copies/μL的病毒,虽低于SYBR GreenⅠ的显色敏感性,但能有效地避免气溶胶造成的空气环境污染。结果表明,本研究建立的荧光显色LAMP体系敏感性高、能避免交叉污染,具有在科研机构、基层实验室特别是检测现场推广和应用的潜力,能够为从源头遏制该病的发生和蔓延提供有效的手段。
Six pairs of loop-mediated isothermal amplification(LAMP)primers were designed according to the conserved region of duck Tembusu virus(DTMUV)E gene retrieved from NCBI.On the basis of optimization of the various components and conditions in the reaction,an LAMP method was developed for detection of DTMUV.Then the fluorescent reagent SYBR GreenⅠand a certain proportion of calcein and manganese ion were used to determined the colour development of products.When SYBR GreenⅠ was used as the fluorescent reagent,the sensitivity was 10copies/μL of the virus,which was 100times higher than that of the conventional PCR.Meanwhile the detection limit of proportion of calcium and manganese ion was 1 000copies/μL.Although the sensitivity of calcein and manganese ion was lower than that of SYBR GreenⅠ,the contaminated aerosol could be effectively avoided.The fluorescent colour LAMP system established in this study had high sensitivity and could avoid the cross contamination.So this system is of great benefit and potential in research institutions,grass-roots laboratories and field test,and can provide effective means to curb the occurrence and spreading of the disease from the sources.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第4期406-411,共6页
Chinese Veterinary Science
基金
公益性行业(农业)科研专项经费项目(201003012)
创新方法专项(2013IM030700)
山东省农业重大应用技术创新课题
关键词
鸭坦布苏病毒
环介导等温扩增
荧光显色剂
duck Tembusu virus
loop-mediated isothermal amplification
fluorescent reagent
