重组酶辅助扩增结合CRISPR/Cas13a检测禽腺病毒4型方法的建立  

Detection of Fowl Adenovirus Serotype 4 by Recombinase Aided Amplification Combined with CRISPR/Cas13a

作　　者：殷冬冬 郭浩 兰梦蝶 尹磊 王洁茹 沈学怀[1,2] 戴银 潘孝成[1,2] YIN Dongdong;GUO Hao;LAN Mengdie;YIN Lei;WANG Jieru;SHEN Xuehuai;DAI Yin;PAN Xiaocheng(Livestock and Poultry Epidemic Diseases Research Center of Anhui Province,Institute of Animal Husbandry and Veterinary Science,Anhui Academy of Agricultural Science,Hefei 230001,China;Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety Engineering,Hefei 230001,China;Animal Health Supervision Institute of Agriculture and Rural Bureau of Feixi County,Hefei 231200,China;Animal Health Supervision Institute of Ningguo City,Ningguo 242300,China)

机构地区：[1]安徽省农业科学院畜牧兽医研究所,安徽省畜禽疫病研究中心,安徽合肥230001 [2]畜禽产品安全工程安徽省重点实验室,安徽合肥230001 [3]肥西县农业农村局动物卫生监督所,安徽合肥231200 [4]宁国市动物卫生监督所,安徽宁国242300

出　　处：《云南农业大学学报（自然科学版）》2023年第5期803-807,共5页Journal of Yunnan Agricultural University:Natural Science

基　　金：安徽省科技重大专项(202003a06020012);安徽省重点研究与开发计划(202204c06020039);财政部和农业农村部现代农业产业技术体系(CARS-40)。

摘　　要：【目的】建立高效、灵敏、特异的禽腺病毒4型(fowl adenovirus serotype 4,FAdV-4)检测方法。【方法】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(clustered regularly interspaced short palindromic repeats associated protein 13a,CRISPR-Cas13a)技术相结合,针对FAdV-4 Hexon基因保守区设计RAA引物及CRISPR RNA(crRNA),利用RAA技术扩增样本核酸,并进行CRISPR-Cas13a荧光检测,以qPCR为对照方法,评价所建立方法的灵敏度、特异性以及与qPCR法的一致性。【结果】本研究所建立的方法反应过程均在37℃恒温条件下完成,反应体系可检测的最低扩增拷贝数为101 copies/μL,灵敏度高,且与FAdV-1、FAdV-7、FAdV-8b、FAdV-9、FAdV-10、鸡传染性喉气管炎病毒、传染性法氏囊病毒、传染性支气管炎病毒、禽流感H9亚型疫苗和新城疫病毒等禽病原核酸检测无交叉反应。该方法检测30份临床样本与qPCR检测的阳性检出率一致。【结论】建立的RAA-Cas13a方法检测FAdV-4具有简便、灵敏、特异等特点,可用于FAdV-4的快速检测和流行病学监测。[Purpose]To establish an efficient,sensitive and specific detection method for fowl adenovirus serotype 4(FAdV-4).[Methods]Recombinase aided amplification(RAA)was used in combination with the clustered regularly interspaced short palindromic repeats associated protein 13a(CRISPR-Cas13a)to design RAA primers and CRISPR RNA(crRNA)targeting the conserved region of the FAdV-4 Hexon gene.The RAA technology was employed to amplify the sample nucleic acid,followed by CRISPR-Cas13a fluorescence detection.The established method was evaluated for sensitivity,specificity,and consistency compared to the qPCR control method.[Results]The method established in this study was performed at 37℃with a minimum amplification copy number of 101 copies/μL,it was sensitive and had no cross-reactivity with avian-derived pathogens such as FAdV-1,FAdV-7,FAdV-8b,FAdV-9,FAdV-10,avian infectious laryngotracheitis virus,infectious bursal disease virus,infectious bronchitis virus,avian influenza subtype H9,newcastle disease virus.The detection rate of 30 clinical samples by this method was consistent with that of qPCR.[Conclusion]The RAA-Cas13a method is simple,sensitive and specific for the detection of FAdV-4,and can be used for rapid detection and epidemiological surveillance of FAdV-4.

关 键 词：禽腺病毒4型 CRISPR/Cas13a 重组酶辅助扩增 检测 

分 类 号：S852.657[农业科学—基础兽医学]