[1]高晓艺,韩　焘,王乃迪,等.H5和H7亚型高致病性禽流感病毒新型冻干微芯片双重荧光定量RT-PCR方法的建立和初步应用[J].中国预防兽医学报,2019,(09):911-916.[doi:0.3969/j.issn.1008-0589.201812005]
　GAO Xiao-yi,HAN Tao,WANG Nai-di,et al.Establishment and application of lyophilized microchip duplex real-time RT-PCR assay for identification of H5 and H7 subtype highly pathogenic influenza viruses[J].Chinese journal of preventive veterinary medicine,2019,(09):911-916.[doi:0.3969/j.issn.1008-0589.201812005]
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H5和H7亚型高致病性禽流感病毒新型冻干微芯片双重荧光定量RT-PCR方法的建立和初步应用()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年09
页码:
911-916
栏目:
诊断和检测技术
出版日期:
2019-10-25

文章信息/Info

Title:
Establishment and application of lyophilized microchip duplex real-time RT-PCR assay for identification of H5 and H7 subtype highly pathogenic influenza viruses
文章编号:
1008-0589(2019)09-0911-07
作者:
 

高晓艺1; 2; 韩　焘1; 王乃迪1; 王传彬1; 杨　林1; 王新杰3; 高姗姗3; 孙晓明3; 胡祥钰3; 石玉祥2*; 刘玉良1*

 (1. 中国动物疫病预防控制中心，北京 102618；2. 河北工程大学 生命科学与食品工程学院，河北 邯郸 056021；
3. 北京亿森宝生物科技有限公司，北京 100025)
Author(s):
 

GAO Xiao-yi1; 2;  HAN Tao1;  WANG Nai-di1;  WANG Chuan-bin1;  YANG Lin1;  WANG Xin-jie3;  GAO Shan-shan3;  SUN Xiao-ming3;  HU Xiang-yu3;  Shi Yu-xiang2*;  Liu Yu-liang1*

 (1. China Animal Disease Control Center, Beijing 102618, China; 2. College of Life Sciences and Food Engineering,
Hebei University of Engineering, Handan 056021, China; 3. Beijing Yisen Biotechnology Co., Ltd., Beijing 100025, China)
关键词:
H5亚型禽流感; H7亚型禽流感; HA裂解位点; 双重荧光定量RT-PCR; 微芯
Keywords:
 ; H5 subtype avian influenza;  ;  H7 subtype avian influenza;  ;  HA cleavage site;  ;  duplex real-time RT-PCR;  ;  microchip
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201812005
文献标志码:
A
摘要:
为建立鉴别检测H5和H7亚型高致病性禽流感病毒(HPAIV)双重荧光定量RT-PCR检测方法，本研究根据已登录的H5和H7亚型HPAIV HA基因裂解位点序列差异分别设计引物和探针，对反应体系各条件优化后首次建立了同时鉴别检测H5与H7亚型HPAIV的新型冻干微芯片双重荧光定量RT-PCR方法。特异性试验结果显示，该方法除对H5和H7亚型HPAIV检测结果为阳性外，对其它几种常见的禽病病原检测结果均为阴性，该方法特异性强；敏感性试验结果显示，该方法检测下限为病毒浓度1×101 TCID50/mL，而常规荧光定量RT-PCR检测下限为1×102 TCID50/mL，敏感性高；标准曲线分析后结果显示，本研究所建立方法的扩增效率比常规荧光定量RT-PCR高，二者相关系数均为0.997；批内和批间重复性试验结果显示，Ct值的变异系数均小于2 %以下，重复性高。临床样品检测结果显示，该方法所需样本微量仅为1 μL～2 μL；操作简便、快速，检测过程<40 min，可用于现场检测；而且利用该方法与常规荧光定量PCR方法以及测序对临床样品进行检测，三者结果完全吻合。本研究所建立的新型冻干微芯片双重荧光定量RT-PCR检测方法为H5和H7亚型HPAIV监测和临床检测提供了可行的技术手段。
　　
Abstract:
To identify H5 and H7 subtype highly pathogenic avian influenza viruses (HPAIVs), a lyophilized microchip duplex real-time RT-PCR (RT-qPCR) assay was initially developed basing on the different reference sequences of the HA cleavage sites of H5 and H7 HPAIVs. Our results revealed that the novel approach has advantages over conventional real-time RT-PCR. The assay was highly specific with positive results in the detection of H5 and H7 HPAIVs and negative results in the detection of the other avian pathogenic agents. This assay showed sensitive with the detection limit (1×101 TCID50/mL), while the detection limit of the conventional real-time RT-PCR method was 1×102 TCID50/mL. This method exhibited higher amplification efficiency than the conventional real-time RT-PCR method with the coefficient of correlation (0.997), and good repeatability with the coefficient of variation of Ct values lower than 2% among repeated testing. The detection by this assay could be completed within 40min, indicating this method was rapid and suitable for on-site detection. In addition, only a tiny volume of 1 to 2μL of sample was needed for testing. A total of 30 clinical samples were detected by this method and the result was consistent with that obtained by conventional RT-PCR. In conclusion, this method we established in this study could be feasible approach for simultaneous detection and identification of H5 and H7 HPAIVs in large-scale surveillance and clinical diagnosis.

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(本文编辑：彭永刚；英文编辑：曾显营)

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更新日期/Last Update: 2019-10-28