不同分子生物学方法在H5亚型禽流感病毒检测中的应用比较  

Comparison of Different Molecular Biotechnologies for Detection of H5-AIV

作　　者：尚佳静 罗娟 于晓慧 房立春 亓丽红[3] 孟鸽 封莹洁 蒋文明[2] 刘华雷[2] 刘冠慧 李阳 Shang Jiajing;Luo Juan;Yu Xiaohui;Fang Lichun;Qi Lihong;Meng Ge;Feng Yingjie;Jiang Wenming;Liu Hualei;Liu Guanhui;Li Yang(College of Life Sciences and Food Engineering,Hebei University of Engineering,Handan,Hebei 056038,China;China Animal Health and Epidemiology Center,Qingdao,Shandongg266032,China;Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan,Shandong 250199,China)

机构地区：[1]河北工程大学生命科学与食品工程学院,河北邯郸056038 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]山东省农业科学院畜牧兽医研究所,山东济南250199

出　　处：《中国动物检疫》2023年第11期100-106,共7页China Animal Health Inspection

基　　金：2023年黄河流域协同科技创新(科技对口支援和东西部协作)项目(YDZX2023009)。

摘　　要：H5亚型高致病性禽流感(H5-HPAI)是严重危害家禽养殖业和公共卫生健康的一类动物疫病,也是我国高度重视、严格防控的人兽共患病。目前常用检测H5亚型禽流感病毒(H5-AIV)的分子生物学技术包括反转录聚合酶链式反应(RT-PCR)、实时荧光定量PCR(RT-qPCR)、反转录重组酶辅助扩增(RT-RAA)、RT-RAA结合侧流层析试纸条(RT-RAA-LFD)、RT-RAA-簇状规则间隔短回文重复序列及其相关蛋白结合侧流层析试纸条(RT-RAA-CRISPRCas13a-LFD)等。为比较不同检测方法对H5-AIV检测的灵敏度差异,将制备的cRNA标准品进行倍比稀释,分别用RT-PCR、RT-qPCR、RT-RAA、RT-RAA-LFD、RT-RAA-CRISPRCas13a-LFD进行检测。结果显示:RT-PCR、RT-qPCR、RT-RAA、RT-RAA-LFD与RT-RAA-CRISPRCas13a-LFD方法的检测灵敏度分别为10^(2)、10^(-1)、10^(0)、10^(0)、10^(-1)copies/μL。与RT-qPCR相比,RT-PCR、RT-RAA、RT-RAA-LFD、RT-RAA-CRISPRCas13a-LFD在临床样本检测上具有较高的符合率,分别为85.00%、93.75%、88.75%、91.25%。结果表明,5种方法均对H5-AIV具有良好的检出率,但RT-qPCR与RT-RAA-CRISPRCas13a-LFD灵敏度更高,分别适合在实验室与基层养殖场进行临床检测。本研究评估了不同方法在H5-AIV检测中的应用效果,为在不同场景下选择准确可靠的检测方法提供了参考,也为H5-AIV感染的防控提供了技术支撑。H5 subtype highly pathogenic avian influenza(H5-HPAI)is one of the category I animal diseases that seriously endangers poultry industry and public health,and is also one of the zoonotic diseases highly concerned and controlled in China.At present,common molecular biological techniques include reverse transcription-polymerase chain reaction(RT-PCR),real-time quantitative PCR(RT-qPCR),reverse transcription-recombinase-aided amplification(RT-RAA),RT-RAA with lateral flow dipstick(RT-RAA-LFD),RT-RAA clustered regularly interspaced short palindromic repeat and their associated proteins combined with lateral flow chromatography test strips(RT-RAA-CRISPR Cas13a-LFD),etc.In order to compare the sensitivity difference of different assays for H5-AIV,the prepared cRNA standards were diluted and detected by RT-PCR,RT-qPCR,RT-RAA,RT-RAA-LFD and RTRAA-CRISPR Cas13a-LFD,respectively.The results revealed that the sensitivity of RT-PCR,RT-qPCR,RT-RAA,RT-RAA-LFD and RT-RAA-CRISPR Cas13a were 10^(2),10^(-1),10^(0),10^(0),and 10'copies/μL,respectively.RT-PCR,RT-RAA,RT-RAA-LFD,RT-RAA-CRISPR Cas13a-LFD were highly compliant in detection of clinical samples when compared with RT-qPCR,which were 85.00%,93.75%,88.75%and 91.25%,respectively.In conclusion,the five methods could detect H5-AIV with a high detection rate,but RT-qPCR and RT-RAA-CRISPR Cas13a-LFD were more sensitive and suitable for clinical test in laboratories and grassroots farms,respectively.The application effectiveness of different detection methods for H5-AIV were evaluated in the study,providing a reference for selecting an accurate and reliable method under different conditions,and a technical support for future prevention and control of H5-AIV infection.

关 键 词：禽流感病毒 H5亚型 检测方法 灵敏度 

分 类 号：S855.3[农业科学—临床兽医学]