猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用  

Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a

作　　者：刘华[1,2] 殷冬冬 邵颖[1] 宋祥军 王振宇 潘孝成[3] 涂健[1] 何长生[2] 朱良强[2] 祁克宗[1] LIU Hua;YIN Dongdong;SHAO Ying;SONG Xiangjun;WANG Zhenyu;PAN Xiaocheng;TU Jian;HE Changsheng;ZHU Liangqiang;QI Kezong(Anhui Agricultural University,Hefei 230036,China;Anhui Animal Disease Prevention and Control Center,Hefei 230091,China;Institute of Animal Husbandry and Veterinary Medicine,Anhui Academy of Agricultural Sciences,Hefei 230031,China)

机构地区：[1]安徽农业大学,合肥230036 [2]安徽省动物疫病预防与控制中心,合肥230091 [3]安徽省农业科学院畜牧兽医研究所,合肥230031

出　　处：《畜牧兽医学报》2023年第9期3991-3997,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金面上项目(31972642);安徽省高校协同创新项目(GXXT-2019-035)。

摘　　要：将重组酶辅助扩增技术(recombinase aided amplification,RAA)与规律间隔性成簇短回文重复序列相关Cas13a蛋白(CRISPR-Cas13a)技术相结合,即RAA-Cas13a,建议建立高效、灵敏、特异的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)检测方法。针对PEDV N基因保守区设计RAA特异性引物和CRISPR RNA(crRNA),利用RAA技术扩增样本核酸,并进行CRISPR-Cas13a荧光检测,以RT-qPCR为对照方法,评价该方法的灵敏度、特异性及与RT-qPCR法的一致性。结果表明,该方法最低可检测至101 copies·μL^(-1),且与猪圆环病毒1型、猪圆环病毒2型、猪圆环病毒3型、猪繁殖与呼吸综合征病毒、猪瘟病毒及猪伪狂犬病病毒等常见猪源病原核酸检测无交叉反应。采用RAA-Cas13a检测40份临床样本与RT-qPCR方法阳性符合率为100%,阴性符合率为84.6%,总符合率为95%,Kappa值为0.881。本研究建立的RAA-Cas13a方法灵敏度高、特异性强,为PEDV的临床诊断和流行病学监测提供了可靠的技术手段。The aim of this paper is to establish a rapid,sensitive,and specific detection method for porcine epidemic diarrhea virus(PEDV)by recombinase aided amplification(RAA)combined with the Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a(CRISPR-Cas13a),called RAA-Cas13a.The specific primer for RAA and CRISPR RNA(crRNA)of the conserved region of the PEDV N gene were designed.The sample nucleic acids were amplified by RAA,whose amplification products were then detected with CRISPR-Cas13a and use RT-qPCR as a control method to evaluate the sensitivity,specificity,and consistency of this method.The results showed that the method could detect as low as 101 copies·μL^(-1) and had no cross-reactivity with pig-derived pathogens such as porcine circovirus type 1,porcine circovirus type 2,porcine circovirus type 3,porcine reproductive and respiratory syndrome virus,swine fever virus and pseudorabies virus.The positive coincidence rate of 40 clinical samples detected by RAA-Cas13a and RT-qPCR method was 100%,the negative coincidence rate was 84.6%,the total coincidence rate was 95%,and the Kappa value was 0.881.The established RAA-Cas13a detection method has high sensitivity and strong specificity,providing a reliable technical means for the clinical diagnosis and epidemiological monitoring of PEDV.

关 键 词：猪流行性腹泻病毒 CRISPR/Cas13a 重组酶辅助扩增 N基因 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]