禽流感病毒与新城疫病毒多靶标核酸质谱检测方法研究及应用  

Studies and Application of Multi-target Nucleic Acid Mass Spectrometry Detection Method for Avian Influenza/Newcastle Disease Virus

作　　者：高志强 赖平安 宋悦谦 种焱 郭悠然 白子龙 郭惠民 汪琳 蒲静 史喜菊 任彤 赵相鹏 GAO Zhiqiang;LAI Ping’an;SONG Yueqian;CHONG Yan;GUO Youran;BAI Zilong;GUO Huimin;WANG Lin;PU Jing;SHI Xiju;REN Tong;ZHAO Xiangpeng(Science and Technology Research Center of China Customs,Beijing 100026,China;Zhejiang Digena Diagnostic Technology Co.Ltd,Hangzhou 311100,China)

机构地区：[1]中国海关科学技术研究中心,北京100026 [2]浙江迪谱诊断技术有限公司,杭州311100

出　　处：《畜牧兽医学报》2025年第3期1386-1395,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划(2021YFF0703804);海关总署科研项目(2022HK125)。

摘　　要：禽流感和新城疫是全球养禽业最具威胁的两种病毒性传染病,快速准确地病原检测和分型对于这两种动物疫病的早期防控和阻断传播具有至关重要的作用。本研究通过将微量多重RT-PCR、单碱基延伸和基于MALDI-TOF的质谱技术联合使用,针对禽流感和新城疫病毒建立了一种高通量核酸质谱检测方法,并对方法的灵敏度、特异性和重复性进行了评价。结果表明,建立的方法可即时同步检测禽流感病毒(A型通用)、H5、H7、H9亚型,新城疫病毒及其中强毒株,各个靶标的检测灵敏度为8.82~15.40 copies·μL^(-1),与其他禽病病原核酸无交叉反应,对12 copies·μL^(-1)的6种混合靶标的检测重复性均在75%以上。对179份实验室收集的送检样品(包括拭子样品和组织脏器)检测结果表明,与荧光RT-PCR检测方法相比,禽流感病毒通用检测靶标符合率为98.9%;其他靶标的符合率均为100.0%。本研究为禽流感、新城疫病毒的快速鉴别检测及重要亚型/株型的同步快速分型鉴定提供了一种新的高通量替代方法。Avian influenza(AI)and Newcastle disease(ND)are the two most threatening viral infectious diseases in the global poultry industry.Rapid and accurate pathogen detection and typing are of vital importance for the early control and transmission blocking of these two animal diseases.(Method)In this study,a high-throughput nucleic acid mass spectrometry method for detection of AIV and NDV was developed by combining micro multiplex RT-PCR,single base extension and MALDI-TOF mass spectrometry,and the sensitivity,specificity and repeatability of the method were evaluated.The results showed that the established method could detect AIV(type A),subtype H5,subtype H7,subtype H9,NDV and its velogenic/mesogenic strains synchronously.The analytical sensitivity of targets was 8.82-15.40 copies·μL^(-1),and there was no cross-reaction with other avian pathogen nucleic acid.The reproducibility of 6 mixed targets at 12 copies·μL^(-1)was above 75%.The detection results of 179 samples(including swab samples and tissue organs)collected in laboratory showed that the coincidence rate of universal target of AIV was 98.9%,and the other targets were 100.0%compared with real-time RT-PCR.So a new high-throughput alternative method is provided for differential detection of AIV,NDV,and their important subtypes/strains synchronously in this study.

关 键 词：禽流感病毒 新城疫病毒 多靶标 核酸质谱 

分 类 号：S851.34[农业科学—预防兽医学]