牛羊赤羽病病毒环介导等温扩增检测方法的建立

Development of a loop-mediated isothermal amplification assay for the detection of bovine and ovine Akabane virus

为建立一种能够快速、简便地检测牛、羊赤羽病病毒的分子生物学方法,根据赤羽病病毒特异性S基因的6个特异区域设计了2对引物,首次建立了赤羽病病毒环介导等温扩增(LAMP)检测方法,对LAMP反应条件进行了优化,并将其与普通PCR方法进行比较。结果显示,LAMP检测方法的特异性好,只对赤羽病病毒进行扩增,且操作简便。本研究确定的最佳反应温度为63℃,核酸检测的最低检出限为1.08ng/μL,比普通PCR的灵敏度高2个数量级。该方法为赤羽病病毒的一种最新的快速、简便的检测方法,可用于出入境牛、羊赤羽病的快速检测。

To establish a molecular biological method for easy and quick detection of bovine and ovine Akabane virus（AKAV）,two pairs of primers were designed according to 6 specific regions of AKAV S gene,and a loop-mediated isothermal amplification（LAMP） assay was established for the first time for the detection of AKAV. Amplification temperature was optimized,and the specificity,the sensitivity,and the reproducibility of the LAMP assay were performed. Electrophoresis revealed that ladder-like products were obtained from the AKAV DNA by the LAMP at 63℃,while no ladder-like products was found from control samples. The detection limit of the LAMP was 1.08ng/μL（DNA）,which was more sensitive than that of traditional PCR method. The LAMP assay established in the present study was quick,simple and original for the detection of AKAV,and could be used for the detection of bovine and ovine Akabane disease in export and import quarantine.