H9和H6亚型禽流感病毒二重RT-PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.12.003

Abstract

Abstract:

This experiment was aimed to develop a method for simultaneous detection of H9 and H6 subtype avian influenza virus (AIV).Two pairs of specific primers were designed according to the conserved regions sequences of H6 and H9 AIV HA gene,a duplex RT-PCR simultaneous detection of H9 and H6 subtype AIV was developed by optimizing the PCR system such as the concentration of different primers and annealing temperature.It showed that all samples could be amplified specific bands from H9 subtype AIV single infection samples or H6 subtype AIV single infection samples,and the samples mix infection these two subtypes AIV.No specific bands of the same sizes were amplified from genomic materials of other avian pathogens.The detection limit of the duplex RT-PCR was 5×10⁴ copies/μL. It suggested that this duplex RT-PCR assay was a specific,sensitive,stable and repeatable method for detection of H9 and H6 subtype of AIV,and could provide technical support for the monitoring of H9 and H6 subtype AIV.

Key words: avian influenza virus; H9 subtype; H6 subtype; duplex RT-PCR

CLC Number:

S852.65

LI DAN, XIE Zhi-xun, SONG De-gui, LI Meng, XIE Zhi-qin, LUO Si-si, XIE Li-ji, HUANG Li, FAN Qing, HUANG Jiao-ling. Development of a Duplex RT-PCR Assay for Detection of H9 and H6 Subtype AIV[J]. , 2016, 43(12): 3101-3106.

References

[1] Tong S, Zhu X, Li Y, et al. New world bats harbor diverse influenza A viruses[J]. PLoS Pathog, 2013, 9(10):e1003657.
[2] Webster R G, Air G M, Metzger D W, et al. Antigenic structure and variation in an influenza virus N9 neuraminidase[J]. J Virol,1987,61(9):2910-2916.
[3] Xie Z, Xie L, Zhou C, et al. Complete genome sequence analysis of an H6N1 avian influenza virus isolated from Guangxi pockmark ducks[J].J Virol,2012, 86(24):13868-13869.
[4] 罗思思,谢芝勋,谢志勤,等. H6N1亚型禽流感病毒二重荧光RT-PCR检测方法的建立[J]. 中国畜牧兽医,2016,43(2):326-332.
[5] 甘孟侯. 禽流感[M]. 北京:北京农业大学出版社, 2002.
[6] Liu D, Shi W, Shi Y,et al. Origin and diversity of novelavian influenza A H7N9 viruses causing human infection:Phylogenetic, structural, and coalescent analyses[J]. Lancet,2013, 381(9881):1926-1932.
[7] Chen H, Yuan H, Gao R,et al. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection:A descriptive study[J]. Lancet, 2014, 383(9918):714-721.
[8] Liu D, Shi W, Gao G F. Poultry carrying H9N2 act as incubators for novel human avian influenza viruses[J]. Lancet, 2014, 383(9920):869.
[9] Xu W, Li X, Bai T, et al. A fatal case of infection with a further reassortant, highly pathogenic avian influenza (HPAI) H5N6 virus in Yunnan, China[J]. Infect Genet Evol, 2016, 40:63-66.
[10] Chin P S,Hoffmann E, Webby R, et al. Molecular evolution of H6 influenza viruses from poultry in Southeasterm China:prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97(H5N1)-like genes in poultry[J]. J Virol, 2002, 76(2):507-516.
[11] Yuan J, Zhang L, Kan X Z, et al. Origin and molecular characteristics of a novel 2013 avian influenza A(H6N1) virus causing human infection in Taiwan[J].Clin Infect Dis, 2013,57(9):1367-1368.
[12] 陈妍梅, 葛万运, 黄川, 等. 广西健康青年H9、H6亚型禽流感病毒血清抗体调查[J]. 中国热带医学, 2008,6:985-986.
[13] 梁庆, 李景东, 陈煜生, 等. 潮汕地区健康青年H9、H6、H5亚型甲型流感病毒血清抗体调查[J]. 汕头大学医学院学报, 2003, 2:107-108.
[14] 罗思思,谢芝勋,谢志勤,等. H6N1亚型禽流感病毒二重RT-PCR检测方法的建立[J]. 畜牧与兽医,2015,47(8):24-27.
[15] 刘志杰,李如举,曾智勇,等. 多重PCR反应的影响因素及其优化[J]. 黑龙江畜牧兽医,2011,13:26-28.
[16] Peng Y,Xie Z X, Liu J B, et al. Epidemiological surveillance of low pathogenic avian influenza virus (LPAIV) from poultry in Guangxi province, Southern China[J].PLoS One, 2013, 8(10):e77132.
[17] Hoffmann E, Stech J, Guan Y, et al. Universal primer set for the full-length amplification of all influenza A viruses[J]. Arch Virol, 2001, 146(12):2275-2289.
[18] Fusaro A, Monne I, Salviato A, et al.Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications[J]. J Virol, 2011, 85(16):8413-8421.
[19] 范胜涛. 野鸟禽流感监测及哺乳动物跨种传播能力评估[D].北京:北京协和医学院,2014.
[20] 赵国,刘晓文,钱忠明,等. 2002-2009年中国华东地区家禽低致病性禽流感的病原学检测与分析[J].中国农业科学,2011,44(1):153-159.
[21] 刘婷婷,谢芝勋,宋德贵,等. H3亚型禽流感病毒巢式PCR检测方法的建立[J].中国畜牧兽医,2014,41(11):85-89.
[22] 徐倩,谢芝勋,谢丽基,等. 禽流感病毒H9N2亚型三重PCR检测方法的建立[J]. 中国畜牧兽医,2014,41(11):58-62.

Development of a Duplex RT-PCR Assay for Detection of H9 and H6 Subtype AIV

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