H5、H7和H9亚型禽流感病毒四重荧光RT-PCR检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2020.026

摘要/Abstract

摘要： 旨在提高对禽流感病毒（avian influenza virus，AIV）检测效率，及时发现疫病。本研究通过分析M基因以及H5、H7和H9亚型的HA基因序列保守区域，设计并合成了相关探针和引物，建立了禽流感病毒（AIV）四重荧光RT-PCR检测方法，该方法可在检测禽流感病毒（AIV）的同时，确定病原是否为H5、H7和H9亚型。结果显示，该检测方法耗时短、特异性好、检测下限达到10^-5 ng·μL^-1。采用该方法检测临床采集的13个活禽交易市场的384份禽咽喉和泄殖腔双拭子样品，经检测，其中有60份样品为流感病毒阳性，且全部是H9亚型，该结果与行业标准方法（NY/T 772—2013）检测结果一致，κ值为1（P<0.001）。本方法能实现对禽流感病毒及H5、H7和H9亚型的高通量快速检测，将在AIV快速检测中发挥重要作用。

关键词: 禽流感, H5亚型, H7亚型, H9亚型, 四重荧光RT-PCR检测方法

Abstract: To shorten the detection time, a quadruple fluorescence RT-PCR method was established for detecting avian influenza virus with the related probes and primers designed and synthesized by analyzing the conservative sequences of M gene, HA gene of H5 subtype, H7 subtype and H9 subtype. This method can detect avian influenza virus, as well as H5 subtype, H7 subtype, and H9 subtype simultaneously. The results showed that the method was quick, specific, and the detection limitation was 10^-5 ng·μL^-1. Sixty avian influenza viruses were detected from 384 chicken swab samples from 13 living birds markets with the method. All the detected avian influenza viruses were H9 subtype. The results were consistent with the method reported in standard, NY/T 772-2013. The κ value for these two methods was 1 (P<0.001). This method can realize the detection of avian influenza virus which has the characteristics of safety, specificity, rapidity, sensitivity, simplicity and high throughput. It will play an important role in the rapid detection of avian influenza virus.

Key words: avian influenza virus, H5 subtype, H7 subtype, H9 subtype, quadruple real-time fluorescence RT-PCR

中图分类号:

S852.659.5

王素春, 仲焕香, 姜楠, 姜利建, 潘子豪, 孙福亮, 刘华雷, 黄保续, 王楷宬. H5、H7和H9亚型禽流感病毒四重荧光RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2020, 51(6): 1429-1437.

WANG Suchun, ZHONG Huanxiang, JIANG Nan, JIANG Lijian, PAN Zihao, SUN Fuliang, LIU Hualei, HUANG Baoxu, WANG Kaicheng. Establishment of the Quadruple Real-time Fluorescence RT-PCR for Detection of H5, H7 and H9 Subtypes Avian Influenza Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1429-1437.

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