高通量核酸液相芯片检测法对H5、H9亚型禽流感病毒及新城疫病毒的检测  

Detection of H5,H9 Subtype AIV and NDV By High Throughput Nucleic Acid Liquid Chip

作　　者：张婷 谢晋雄 詹爱军 吴雯娟 黄彬庚 吴绍精 陈勇 ZHANG Ting;XIE Jinxiong;ZHAN Aijun;WU Wenjuan;HUANG Bingeng;WU Shaojing;CHEN Yong(Shenzhen Academy of Inspection and Quarantine,Shenzhen,Guangdong 518010;Shenzhen Agricultural Product Quality Safety Inspection and Testing Center,Shenzhen,Guangdong 518055;Xinyu Agricultural Science Research Institute,Xinyu,Jiangxi 338025)

机构地区：[1]深圳市检验检疫科学研究院,广东深圳518010 [2]深圳市农产品质量安全检验检测中心,广东深圳518055 [3]新余市农业科学研究所,江西新余338025

出　　处：《中国家禽》2018年第16期18-24,共7页China Poultry

基　　金："十二五"支撑项目(2012BAK17B08);国家质检总局项目(2015IK245)

摘　　要：为建立禽流感病毒和新城疫病毒的液相芯片快速检测方法,试验设计并合成针对H5、H9亚型禽流感病毒HA基因以及新城疫病毒NP基因的特异性引物、探针及探针反向序列,并将下游引物和探针反向序列进行生物素标记。将探针与不同编号的荧光编码微球偶联。通过将探针-荧光编码微球偶联物与HA、NP基因的PCR产物杂交反应,用液相芯片检测仪检测荧光信号建立禽流感和新城疫快速液相芯片检测方法。结果显示,该法具有较高的特异性和灵敏度,非特异性反应很小,无明显交叉反应,该液相芯片快速高通量检测方法可同时检测新城疫病毒、H9亚型和H5亚型禽流感病毒,三株病毒毒株抗原的检测灵敏度分别为10^(-4)、10^(-5)和10^(-5)。同时,检测结果显示:三个毒株同步检测与单个毒株分别检测在检测的灵敏上度基本相当。研究建立的可以同时检测新城疫病毒、H9亚型和H5亚型禽流感病毒的快速高通量液相芯片方法,为该类病毒的检测提供了一种新工具,同时也为其他类似病毒的检测提供了借鉴。In this study a rapid method for the detection of avian influenza virus（AIV） and Newcastle disease virus（DNV） by high throughput nucleic acid liquid chip was established. Specific probes and reverse sequence for HA gene of H5, H9 subtype AIV and NP gene of NDV were designed and synthesized respectively, and the downstream primers and probe reverse sequence with biotin were labeled. The labeled probes were coupled with fluorescent beads encoded with different numbers. Then, the probe labled microspheres were hybridized with the PCR products of HA and NP genes. By using liquid chip detector to detect fluorescence signals, a rapid liquid chip method for detection of AIV and NDV was established. The test results showed that the method had high specificity and sensitivity, non-specific reaction was very small, and no obvious cross reaction. The rapid and high-throughput method for detection of NDV,H9 subtype AIV and H5 subtype AIV could be detected by the liquid chip method, and the sensitivity of three strains of virus antigen detected was 10（-4）, 10（-5） and 10（-5） respectively. At the same time, the test results showed that simultaneous detection of three strains and single strain showed the same sensitivity or slightly lower than that of single strain. This study established a rapid high-throughput solution could simultaneously detect NDV, H9 subtype AIV and AIV subtype H5 by chip technology which provided a new tool for the detection of the virus, but also for the detection of other similar viruses.

关 键 词：禽流感病毒 新城疫病毒 液相芯片 

分 类 号：S851.4[农业科学—预防兽医学]