鸭腺病毒3型PCR检测方法的建立
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摘要
鸭3型腺病毒(Duck adenovirus type 3,DAdV-3)为近年来新发的以引起鸭肝脏肿大出血、肾脏大面积出血点为特征的鸭群新发疫病。为建立特异性的检测DAdV-3的PCR方法,研究通过分析鸭群中鸭腺病毒A型(DAdV-A)、鸭源禽腺病毒4型(FAdV-4)、鸭2型腺病毒(DAdV-2)和DAdV-3的Fiber 2基因特征,建立特异性检测DAdV-3的PCR方法。结果显示:优化后的PCR方法最佳退火温度为54℃,对DAdV-3扩增片段大小为548 bp;敏感性强,最低检测限为36.4 pg;特异性好,对DAdV-A、FAdV-4、鸭病毒性肠炎(DEV)、番鸭细小病毒(MDPV)、鹅细小病毒(GPV)、鸭圆环病毒(DuCV)和鹅多瘤病毒(GHPV)均无特异性扩增。表明该方法可有效用于开展新发DAdV-3的流行病学调查。
Duck adenovirus type 3(DAdV-3),which is newly identified adenovirus in ducklings,with necrosis and hemorrhage in liver and spleen.In this study,the Fiber 2 gene sequences of duck adenoviruse A(DAdV-A),fowl adenovirus 4(FAdV-4),duck adenovirus 2(DAdV-2) and DAdV-3 occurred in ducks were retrieved from GenBank in order to develop a specific PCR tool for DAdV-3 infection.Primer design software Oligo 7.0 was used to design specific primers based on the Fiber 2 gene characterization after genetic comparison.Then PCR method was established after conditional optimized.The results showed the optimal annealing temperature was 54 ℃,which only could amplify DAdV-3infection with 548 bp in length.The PCR method showed high sensitivity,with low detection limit at 36.4 pg.The PCR method was demonstrated good specificity,with no cross-amplification from common adenoviruses occurred in ducks(i.e.DAdV-A,FAdV-4) and other common duck-origin pathogens(i.e.duck enteritis virus,Muscovy duck parvovirus,goose parvovirus,duck circovirus and goose hemorrhagic polyomavirus).It′s concluded that PCR method for specific diagnosis with DAdV-3 was developed in this study,which laid foundation for further DAdV-3 epidemiological investigation.
Duck adenovirus type 3(DAdV-3),which is newly identified adenovirus in ducklings,with necrosis and hemorrhage in liver and spleen.In this study,the Fiber 2 gene sequences of duck adenoviruse A(DAdV-A),fowl adenovirus 4(FAdV-4),duck adenovirus 2(DAdV-2) and DAdV-3 occurred in ducks were retrieved from GenBank in order to develop a specific PCR tool for DAdV-3 infection.Primer design software Oligo 7.0 was used to design specific primers based on the Fiber 2 gene characterization after genetic comparison.Then PCR method was established after conditional optimized.The results showed the optimal annealing temperature was 54 ℃,which only could amplify DAdV-3infection with 548 bp in length.The PCR method showed high sensitivity,with low detection limit at 36.4 pg.The PCR method was demonstrated good specificity,with no cross-amplification from common adenoviruses occurred in ducks(i.e.DAdV-A,FAdV-4) and other common duck-origin pathogens(i.e.duck enteritis virus,Muscovy duck parvovirus,goose parvovirus,duck circovirus and goose hemorrhagic polyomavirus).It′s concluded that PCR method for specific diagnosis with DAdV-3 was developed in this study,which laid foundation for further DAdV-3 epidemiological investigation.
引文
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[13]ISMAIL A M,LEE J S,LEE J Y,et al.Adenoviromics:Mining the human adenovirus species D genome[J].Front Microbiol,2018(9):2178.
[14]BOUQUET J F,MOREAU Y,MCFERRAN J B,et al.Isolation and characterisation of an adenovirus isolated from Muscovy ducks[J].Avian Pathol,1982,11(2):301-307.
[15]陈峰,招丽婵,覃健萍,等.应用宏基因组学鉴定国内第一株鸭2型腺病毒[J].中国预防兽医学报,2015(12):903-907.
[16]陈亮,黄路生,刘晓文,等.鸭2型腺病毒的分离鉴定及hexon基因的序列分析[J].中国兽医科学,2016(12):1538-1542.
[2]MAREK A,KAJáN G L,KOSIOL C,et al.Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2 extend the number of species within the genus Aviadenovirus[J].Virology,2014(462-463):107-114.
[3]卢凤英,孙华伟,张敬峰,等.鸭源血清4型禽腺病毒的分离鉴定[J].中国家禽,2018,40(5):62-64.
[4]CHEN H,DOU Y,ZHENG X,et al.Hydropericardium hepatitis syndrome emerged in Cherry Valley ducks in China[J].Transbound Emerg Dis,2017,64(4):1262-1267.
[5]PAN Q,LIU L,WANG Y,et al.The first whole genome sequence and pathogenicity characterization of a fowl adenovirus 4 isolated from ducks associated with inclusion body hepatitis and hydropericardium syndrome[J].Avian Pathol,2017,46(5):571-578.
[6]YU X,WANG Z,CHEN H,et al.Serological and pathogenic analyses of fowl Adenovirus serotype 4(FAdV-4)strain in Muscovy ducks[J].Front Microbiol,2018(9):1163.
[7]ZHANG X,ZHONG Y,ZHOU Z,et al.Molecular characterization,phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014[J].Virology,2016(493):12-21.
[8]周镇海.鸭腺病毒3型基因组序列分析及致病性研究[D].广州:华南农业大学,2016.
[9]WAN C,CHEN C,CHENG L,et al.Development of a TaqMan-based real-time PCR for detecting duck adenovirus 3[J].J Virol Methods,2018(261):86-90.
[10]YIN L,CHEN L,LUO Y,et al.Recombinant fiber-2 protein protects Muscovy ducks against duck adenovirus 3(DAdV-3)[J].Virology,2018(526):99-104.
[11]万春和,刘荣昌,陈翠腾,等.鸭腺病毒A型Hexon基因克隆与序列分析[J].中国畜牧兽医,2017,44(10):3042-3048.
[12]CHEN Z,SHI S,QI B,et al.Hydropericardium syndrome caused by fowl adenovirus serotype 4 in replacement pullets[J].J Vet Med Sci,2018.
[13]ISMAIL A M,LEE J S,LEE J Y,et al.Adenoviromics:Mining the human adenovirus species D genome[J].Front Microbiol,2018(9):2178.
[14]BOUQUET J F,MOREAU Y,MCFERRAN J B,et al.Isolation and characterisation of an adenovirus isolated from Muscovy ducks[J].Avian Pathol,1982,11(2):301-307.
[15]陈峰,招丽婵,覃健萍,等.应用宏基因组学鉴定国内第一株鸭2型腺病毒[J].中国预防兽医学报,2015(12):903-907.
[16]陈亮,黄路生,刘晓文,等.鸭2型腺病毒的分离鉴定及hexon基因的序列分析[J].中国兽医科学,2016(12):1538-1542.