逆转录环介导等温扩增联合横向流动试纸条快速检测草鱼(Ctenopharygodon idellus)呼肠孤病毒(GCRV)的研究

摘要

Grass carp reovirus (GCRV) is a double-chain segment of RNA virus,and can cause outbreak of grass carp hemorrhage in Chines freshwaters.At present,the popular clinical strain of GCRV is in genotype Ⅰ and Ⅱ.To establish a quick and sensitive technology that specific for the detection of GCRV isolations,a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) method was developed.A set of four primers and a labeled probe were designed specifically to recognize six distinct regions respectively to the sixth gene sequence of genotype Ⅰ and Ⅱ GCRV.The method was specific to the detection of GCRV but insusceptible to cyprinid herpesvirus 2 (CyHV-2),spring viremia of carp virus (SVCV),Aeromonas hydrophila (Ah),and other common fish diseases.Moreover the method could detect GCRV of own genotype exclusively in high sensitivity and low detection limit to 1.19 pg for genotype Ⅰ GCRV RNA (2 orders of magnitude greater than that of traditional RT-PCR does) and to 1.88 pg for genotype Ⅱ GCRV RNA (1 order of magnitude greater).The assay could be conducted in one-step amplification at 63℃ in a single tube within 40 min with visualized results by LFD method,and the interpretation from nucleic acid amplification to LFD detection cost only 50 min.Therefore,the method is convenient,quick,sensitive,and GCRV-specific,proving a novel tool to detect GCRV and grass carp hemorrhage.%草鱼呼肠孤病毒(grass carp reovirus,GCRV)是一种双链分节段RNA病毒,是引起草鱼出血病的(hemorrhage of grass carp)的病原,目前临床上较为流行的为基因Ⅰ型和Ⅱ型GCRV.本研究利用逆转录环介导等温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP)进行核酸扩增,通过横向流动试纸条方法(lateral flow dipstick,LFD)进行可视化检测,分别建立了可应用于基因Ⅰ型和Ⅱ型GCRV快速检测的RT-LAMP-LFD技术.该技术以基因Ⅰ型和Ⅱ型GCRV第六基因为检测靶标,分别设计了2对特异性引物(其中,上游内引物由生物素biotin标记)和1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针.结果表明,RT-LAMP最适反应温度为63C,扩增时间为40min,从核酸扩增到LFD结果判读仅需50min.利用RT-LAMP-LFD可特异性检出基因Ⅰ型和Ⅱ型GCRV,而对鲤疱疹病毒Ⅱ型(CyHV-2)、鲤春病毒血症病毒(SVCV)、嗜水气单胞菌等鱼类常见病原的核酸检测结果为阴性,而且两种基因型RT-LAMP-LFD方法只能检测本基因型GCRV,特异性良好.该方法最低可检测到1.19pg的基因Ⅰ型GCRV的RNA,1.88pg基因Ⅱ型GCRV的RNA,其灵敏度分别比相应的RT-PCR方法高2个数量级和1个数量级.对实际样品的检测结果表明,两种基因型RT-LAMP-LFD方法检测GCRV与普通RT-PCR扩增并测序的结果一致.因此,该方法可快速、特异地检测出GCRV,而且操作简单,费用低且耗时短,不需特殊仪器设备,有望成为GCRV现场检测的常规技术手段.