禽流感病毒实时荧光定量RT-PCR检测方法的建立
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摘要
为满足禽流感病毒高通量快速检测的需要,建立了一种能够检测各亚型禽流感病毒的实时荧光定量RT-PCR检测方法,并对该方法的特异性和灵敏度进行评估,使用该方法与农业行业标准NY/T772—2013中的禽流感病毒RT-PCR方法同时进行临床样品检测。结果显示,该方法检测耗时短、特异性好,检测下限达10-4 ng/μL,与传统的RT-PCR方法阳性符合率为100%。结果表明,本方法能实现对禽流感病毒的安全、特异、快速、灵敏、简单、高通量检测,从而弥补了现有传统检测技术的不足。
In order to achieve the high-throughput and rapid detection of avian in?uenza virus(AIV),a real-time fluorescent quantitative RT-PCR method was established for detecting all subtypes of AIV. Then its specificity and sensitivity were evaluated,and some clinical samples were tested simultaneously by the established method and the conventional RT-PCR stipulated in the agricultural industry standard NY/T772—2013. Results showed that the detection limit of the established method could reach 10-4 ng/μL with short detection time and better speci?city,and the positive coincidence rate was 100% as compared with traditional RT-PCR method. In conclusion,the established real-time RTPCR method was safe,speci?c,rapid,sensitive and simple,and could achieve the high-throughput detection for AIV,which covered the de?ciency of current traditional detection technologies.
In order to achieve the high-throughput and rapid detection of avian in?uenza virus(AIV),a real-time fluorescent quantitative RT-PCR method was established for detecting all subtypes of AIV. Then its specificity and sensitivity were evaluated,and some clinical samples were tested simultaneously by the established method and the conventional RT-PCR stipulated in the agricultural industry standard NY/T772—2013. Results showed that the detection limit of the established method could reach 10-4 ng/μL with short detection time and better speci?city,and the positive coincidence rate was 100% as compared with traditional RT-PCR method. In conclusion,the established real-time RTPCR method was safe,speci?c,rapid,sensitive and simple,and could achieve the high-throughput detection for AIV,which covered the de?ciency of current traditional detection technologies.
引文
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[2]TONG S,LI Y,RIVAILLER P,et al.A distinct lineage of influenza A virus from bats[J].Proceedings of the national academy of sciences of USA,2012,109(11):4269-4274.
[3]TONG S,ZHU X,LI Y,et al.New world bats harbor diverse influenza A viruses[J].PLoS pathog,2013,9(10):e1003657.
[4]于乐荣,李小云,汪力斌,等.禽流感发生对家禽养殖农户的经济影响评估:基于两期面板数据的分析[J].中国农村经济,2009,7(1):12-19.
[5]黄泽颖,王济民.高致病性禽流感对我国肉鸡产业的影响[J].中国农业科技导报,2016,18(1):189-199.
[6]CHAN P K.Outbreak of avian influenza A(H5N1)virus infection in Hong Kong in 1997[J].Clinical infectious diseases,2002,34(Suppl 2):58-64.
[7]HAN D D,HAN C X,LI L Y,et al.Epidemiology of human infection with avian influenza A(H7N9)virus in China,2013-2017[J].Zhonghua Liu Xing Bing Xue Za Zhi,2018,39(1):44-46.
[8]HALL B G.Building phylogenetic trees from molecular data with MEGA[J].Molecular biology and evolution,2013,30(5):1229-1235.
[9]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular biology and evolution,2013,30(12):2725-2729.
[10]QIU Y,CHEN J M,WANG T,et al.Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods[J].Virus research,2017,237:22-26.
[11]KIM D W.Real time quantitative PCR[J].Experimental and molecular medicine,2001,33(Suppl 1):101-109.
[12]NAGY A,VOSTINAKOVA V,PIRCHANOVA Z,et al.Development and evaluation of a one-step realtime RT-PCR assay for universal detection of influenza Aviruses from avian and mammal species[J].Archives of virology,2010,155(5):665-673.
[13]SHORTRIDGE K F,ZHOU N N,GUAN Y,et al.Characterization of avian H5N1 influenza viruses from poultry in Hong Kong[J].Virology,1998,252(2):331-342.
[14]GUANY,SMITHGJ.Theemer genceand diversification of panzootic H5N1 influenza viruses[J].Virus research,2013,178(1):35-43.
[15]BI Y,XIE Q,ZHANG S,et al.Assessment of the internal genes of influenza A(H7N9)virus contributing to high pathogenicity in mice[J].Journal of virology,2015,89(1):2-13.