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.
作者
殷冬冬
郭浩
兰梦蝶
尹磊
王洁茹
沈学怀
戴银
潘孝成
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)
出处
《云南农业大学学报（自然科学版）》
CSCD
北大核心
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)。
