基因Ⅱ型草鱼呼肠孤病毒RAA-CRISPR/Cas12a检测方法的建立

ESTABLISHMENT OF RAA-CRISPR/CAS12A DETECTION FOR GENOTYPE Ⅱ GRASS CARP REOVIRUS

为建立一种特异性强、灵敏度高、操作便捷的Ⅱ型草鱼呼肠孤病毒(GCRV-Ⅱ)检测方法,研究利用重组酶介导链置换核酸扩增技术(Recombinase-aid amplification,RAA)与成簇的规律间隔短回文重复序列(CRISPR)系统相结合,构建了GCRV-Ⅱ病毒RAA结合CRISPR/Cas12a可视化“一步”快速检测方法。首先选择GCRV-Ⅱ毒株间序列保守,不同基因型GCRV间差异明显的s6基因(GQ896337)的保守区设计5对RAA候选引物和2对crRNA,合成并筛选出特异性引物和crRNA组合。其次测试反应的特异性、灵敏度和准确性。最后建立下层扩增相、上层检测相的“一步法”反应体系。该检测方法在37℃恒温下反应可检测出GCRV-Ⅱ,最低检测限为10^(2) copies/μL。对24份临床样本进行检测,该检测方法阳性检出率高于现行国标PCR法。研究建立的GCRV-Ⅱ检测方法操作便捷、特异性高、灵敏度好、可重复性强、与设备兼容性好、在蓝光灯下可通过肉眼判断检测结果。

Grass carp reovirus(GCRV)is responsible for severe hemorrhagic disease in grass carp,posing a significant threat to grass carp farming in our country.The type Ⅱ strain of grass carp reovirus(GCRV-Ⅱ)is the primary pathogen causing this disease.In order to establish a specific,sensitive,and easy-to-use detection method for GCRV-Ⅱ,we employed a recombinant enzyme-mediated strand-exchange nucleic acid amplification(RAA)technique.In this study,we combined recombinase-aided amplification(RAA)with the clustered regularly interspaced short palindromic repeats(CRISPR)system to construct a one-step,rapid,and visual detection method for the GCRV-Ⅱ virus.We began by selecting the conserved region of s6 gene(GQ896337),which is consistent among GCRV-Ⅱ strains but significantly different across various GCRV genotypes.We designed five pairs of RAA candidate primers and four crRNAs,synthesizing and screening them to identify the specific primer and crRNA combinations.Subsequently,the specificity,sensitivity,and accuracy of the reaction were evaluated.Finally,a“one-step”reaction system comprised a lower amplification phase and an upper detection phase.The results demonstrated that GCRV-Ⅱ could be detected at a constant temperature of 37℃,with a detection limit as low as 10^(2) copies/μL.When tested on twenty-four clinical samples,the positive detection rate of this method surpassed that of the PCR method currently used in the industry.The detection method for GCRV-Ⅱ established in this study is easy to use,highly specific,sensitive,and reproducible.It is also compatible with existing equipment and allows for visual assessment under blue light.