传染性皮下和造血器官坏死病毒RPA与CRISPRCas12a可视化快速检测方法的建立  

Establishment of rapid visual detection methods for infectious hypodermal and hematopoietic necrosis virus based on RPA and CRISPR-Cas12a

作　　者：周傲白雪 徐博文 沙才华 邵建宏 赵福振 廖秀云 黄海超 蒋晓霞 罗宝正 陈轩 ZHOU Aobaixue;XU Bowen;SHA Caihua;SHAO Jianhong;ZHAO Fuzhen;LIAO Xiuyun;HUANG Haichao;JIANG Xiaoxia;LUO Baozheng;CHEN Xuan(Technology Center of Gongbei Customs District,Zhuhai 519015,China)

机构地区：[1]拱北海关技术中心,广东珠海519015

出　　处：《中国兽医科学》2024年第5期595-601,共7页Chinese Veterinary Science

基　　金：广东省重点领域研发计划项目(2022B1111030001);拱北海关科研项目(2023GK009)。

摘　　要：根据传染性皮下和造血器官坏死病毒(IHHNV)基因序列的保守区域设计并合成多组RPA引物和特异性crRNA,经过筛选与优化,建立RPA-Cas12a系统荧光检测法和试纸条法2种可视化快速检测IHHNV的方法。结果显示,这2种可视化检测方法在37℃恒温反应40 min可检测出IHHNV;与对虾白斑综合征病毒、虾肝肠胞虫和十足目虹彩病毒1无交叉反应;该方法灵敏度较高,最低检测浓度为10 copies/μL;应用该方法对10份IHHNV阳性临床样本进行检测,阳性检出率为100%。综合结果表明,建立的荧光法和试纸条2种可视化检测IHHNV的方法操作简单,反应灵敏,适用于对虾养殖场或基层实验室对IHHNV的定期监测和初筛。After screening and optimization,two visual rapid detection methods,the RPA-Cas12a fluorescent detection and lateral flow dipstick,for infectious hypodermal and hematopoietic necrosis virus(IHHNV)were established by designing and synthesizing multiple sets of recombinase polymerase amplification primers and specific CRISPR RNA based on the conserved regions of the IHHNV gene sequences.The results demonstrated that both visual detection methods could detect IHHNV after a constant temperature reaction at 37℃for 40 min.There was no cross-reactivity with white spot syndrome virus,Enterocytozoon hepatopenaei and decapod iridescent virus 1.The method exhibited high sensitivity,with a detection limit of 10 copies/μL.When applied to test 10 positive clinical samples of IHHNV,the conformity rate was 100%.Overall,the established fluorescence and paper strip visual detection methods are simple to operate,highly sensitive,and suitable for regular monitoring and preliminary screening of IHHNV in shrimp farming facilities or basic laboratories.

关 键 词：传染性皮下和造血器官坏死病毒 重组酶聚合酶扩增 CRISPR-Cas12a 检测方法 

分 类 号：S852.659.5[农业科学—基础兽医学]