摘要
为提高猪Hokovirus(PHoV)检测的敏感性和简便性,本研究采用环介导等温扩增(LAMP)技术,建立了一种敏感、特异、快速、简便、实用的检测PHoV的技术。针对PHoV VP1/2基因保守区设计4条引物,进行LAMP扩增,对扩增反应进行优化并检测其敏感性和特异性。结果表明,所建立的方法最佳反应时间为60 min,反应温度为65℃,该方法具有良好的特异性,与其它常见动物病毒(PRRSV、PCV2、CSFV、PRV、PPV、JEV)无交叉反应,对PHoV的最低检出量为10拷贝,其敏感性是PCR方法的10倍,且通过加入荧光染料可直接判定结果。研究表明,该方法特异性强、敏感性高,且操作简便、检测成本低、检测时间短,不需要特殊的仪器,本研究建立的LAMP方法可以作为PHoV在临床上的一种检测技术。
In this study,a loop-mediated isothermal amplification （LAMP） assay was developed for the rapid,specific and sensitive detec tion of PHoV.A set of four primers specific for six regions within the PHoV VP1/2 genes was designed by using online software.The result showed that the reaction temperature and time of the method were optimized at 65℃C and 60 min,respectively.The method was highly specific for PHoV,and no cross-reaction was observed with porcine reproductive and respiratory syndrome virus （PRRSV）,porcine circovirus type 2 （PCV2）,classic swine fever virus （CSFV）,pseudorabies virus （PRV）,porcine parvovirus （PPV） and Japanese encephalitis virus （JEV）.The detection limit was approximately 10 copies per reaction,10 times more sensitive than conventional PCR,and LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to a fluorescent dye.These results indicated that the LAMP assay was a simple,rapid,sensitive and specific method for detecting PHoV,which did not require any specialized equipment and could be used to detect PHoV both in the laboratory and in the field.
出处
《西南农业学报》
CSCD
北大核心
2014年第2期859-863,共5页
Southwest China Journal of Agricultural Sciences
基金
江苏省农业科技自主创新资金[CX(12)3065]
