[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]
点击复制

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()
分享到:

《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12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-yi12 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.

参考文献/References:

[1]Wu Ying, Wu Yan, Tefsen B, et al. Bat-derived influenza-like viruses H17N10 and H18N11 [J]. Trends Microbiol, 2014, 22 (4): 183-191.
[2]胡志明. 散养鸡高致病性禽流感的防控措施[J]. 中国畜牧兽医文摘,2017,33(8):162.
[3]杜建,兰邹然,翟新验,等. 2017年全球高致病性禽流感疫情分析[J]. 中国动物检疫,2018,35(11):5-8.
[4]蒋文明,李金平,侯广宇,等. H5亚型高致病性禽流感病毒抗原性变异分析[J]. 中国动物检疫,2016,33(09):27-32.
[5]De Jong J C, Claas E C, Osterhaus A D, et al. A pandemic warning? [J]. Nature, 1997, 389(6651): 554.
[6]蒋文明,李金平,彭程,等. 第2.3.4.4分支H5亚型高致病性禽流感病毒主要抗原位点分析[J]. 中国兽医学报,2017,37(11):2121-2125.
[7]Gao Rong-bao, Gao Bin, Hu Yun-wen, et al. Human infection with novel avian-origin influenza A (H7N9) virus [J]. New Engl J Med, 2013, 368(16): 1888-1897.
[8]王霞,何婉婷,李伟强,等. 当前我国H7N9亚型禽流感状况分析[J]. 动物医学进展,2018,39(09):126-129. 
[9]刘东,张竹君,胡娇,等. 2013~2017年我国H7N9亚型禽流感病毒在家禽中的流行情况分析[J]. 病毒学报,2018, 34(06):800-809.
[10]Chang H K, Park J H, Song M S, et al. Development of multiplex RT-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens [J]. J Microbiol Biotechnol, 2008, 18(6): 1164-1169.
[11]王振全,罗宝正,薄清如,等. 基因芯片方法检测6 种动物源性人兽共患病病原[J]. 中国预防兽医学报,2011,33(10):804-807.
(本文编辑:彭永刚;英文编辑:曾显营)

相似文献/References:

[1]齐艳萍,陈雪龙,尹再军,等.白翅浮鸥H5亚型禽流感及新城疫的卵黄抗体监测[J].中国预防兽医学报,2011,(10):824.[doi:10.3969/j.issn.1008-0589.2011.10.17]
 Qi Yan-ping,Chen Xue-long,Yin Zai-jun,et al.Survey of the yolk antibody against H5 subtype avian influenza and Neweastle disease in white-winged tern[J].Chinese journal of preventive veterinary medicine,2011,(09):824.[doi:10.3969/j.issn.1008-0589.2011.10.17]

更新日期/Last Update: 2019-10-28