鸭腺病毒3型MGB TaqMan探针实时荧光定量PCR检测方法的建立
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摘要
鸭(Anas platyrhynchos domestica)腺病毒3型(Duck adenovirus type 3, DAdV-3)是近年来我国新发现的鸭源腺病毒。本研究根据NCBI数据库中DAdV-3代表株(CH-GD-12-2014株, GenBank No. KR135164)的22K基因特征,设计特异性引物和探针,建立检测DAdV-3的MGB TaqMan探针qRT-PCR方法。结果表明,建立的qRT-PCR方法具有如下特性:灵敏度高,最低检测限为55拷贝/μL;特异性强,和鸭群常见传染病病原均无交叉反应;重复性好,组内和组间重复实验变异系数分别为0.44%～2.10%和0.64%～2.37%。利用建立的qRT-PCR方法对临床送检的61份鸭源病料进行检测,检出DAdV-3阳性15份,阳性率为24.6%。本研究为后续开展DAdV-3感染的实验室快速诊断和流行病学调查奠定了初步基础。
Duck(Anas platyrhynchos domestica) adenovirus type 3(DAdV-3) is a newly discovered duckderived adenovirus in recent years. In this study, specific primers and probe were designed based on the 22 K gene characteristics of DAdV-3 representative strain(CH-GD-12-2014 strain, GenBank No. KR135164) in the NCBI database, and a MGB TaqMan probe real-time quantitative PCR method for detecting DAdV-3 was established. The results showed that the established q RT-PCR method had the following characteristics: High sensitivity, minimum detection limit of 55 copies/μL; Strong specificity, and no cross-reaction with common infectious disease pathogens in duck flocks; Good reproducibility, intra-group and inter-group repetitive experimental variation coefficients were 0.44%～2.10% and 0.64%～2.37%, respectively. 61 duck-derived materials were detected by the established method, and 15 positive DAdV-3 samples were detected with a positive rate of 24.6%. This study laid the foundation for rapid laboratory diagnosis and epidemiological investigation of DAdV-3 infection.
Duck(Anas platyrhynchos domestica) adenovirus type 3(DAdV-3) is a newly discovered duckderived adenovirus in recent years. In this study, specific primers and probe were designed based on the 22 K gene characteristics of DAdV-3 representative strain(CH-GD-12-2014 strain, GenBank No. KR135164) in the NCBI database, and a MGB TaqMan probe real-time quantitative PCR method for detecting DAdV-3 was established. The results showed that the established q RT-PCR method had the following characteristics: High sensitivity, minimum detection limit of 55 copies/μL; Strong specificity, and no cross-reaction with common infectious disease pathogens in duck flocks; Good reproducibility, intra-group and inter-group repetitive experimental variation coefficients were 0.44%～2.10% and 0.64%～2.37%, respectively. 61 duck-derived materials were detected by the established method, and 15 positive DAdV-3 samples were detected with a positive rate of 24.6%. This study laid the foundation for rapid laboratory diagnosis and epidemiological investigation of DAdV-3 infection.
引文
周镇海.2016.鸭腺病毒3型基因组序列分析及致病性研究[D].硕士学位论文,华南农业大学,导师:钟仰进,pp.8-63.(Zhou Z H.2016.Genonmic sequence analysis and pathogenecity study of duck adenovirus 3[D].Thesis for M.S.,South China Agricultural University,Suppervisor:Zhong Y J,pp.8-63.)
Ahi Y S,Mittal S K.2016.Components of adenovirus genome packaging[J].Frontiers in Microbiology,7:1503
Biasiotto R,Akusj?rvi G.2015.Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33Kand L4-22K proteins[J].International Journal of Molecular Sciences,16(2):2893-2912.
Bouquet J F,Moreau Y,McFerran J B,et al.1982.Isolation and characterisation of an adenovirus isolated from Muscovy ducks[J].Avian Pathology,11(2):301-307.
Cha S Y,Kang M,Moon O K,et al.2013.Respiratory disease due to current Egg drop syndrome virus in Pekin ducks[J].Veterinary Microbiology,165(3-4):305-311.
Das S,Fearnside K,Sarker S,et al.2017.A novel pathogenic aviadenovirus from red-bellied parrots(Poicephalus rufiventris)unveils deep recombination events among avian host lineages[J].Virology,502:188-197.
Guimet D,Hearing P.2013.The adenovirus L4-22K protein has distinct functions in the posttranscriptional regulation of gene expression and encapsidation of the viral genome[J].Journal of Virology,87(13):7688-7699.
Kaján G L,Stefancsik R,Ursu K,et al.2010.The first complete genome sequence of a non-chicken aviadenovirus,proposed to be turkey adenovirus 1[J].Virus Research,153(2):226-233.
Kang M,Cha S Y,Jang H K.2017.Tropism and infectivity of duck-derived egg drop syndrome virus in chickens[J].PLoS One,12(5):e0177236.
Kutyavin I V,Afonina I A,Mills A,et al.2000.3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures[J].Nucleic Acids Research,28(2):655-661.
Lan S,Kamel W,Punga T,et al.2017.The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding[J].Nucleic Acids Research,45(4):1731-1742.
Li C J,Li H Y,Wang D D,et al.2016.Characterization of fowl adenoviruses isolated between 2007 and 2014 in China[J].Veterinary Microbiology,197():62-67.
Marek A,Kaján G L,Kosiol C,et al.2014.Complete genome sequences of pigeon adenovirus 1 and duck adenovirus2 extend the number of species within the genus Aviadenovirus[J].Virology,462-463:107-114.
Marek A,Ballmann M Z,Kosiol C,et al.2014.Whole-genome sequences of two turkey adenovirus types reveal the existence of two unknown lineages that merit the establishment of novel species within the genus Aviadenovirus[J]The Journal of General Virology,95(1):156-170.
Mittal D,Jindal N,Tiwari A K,et al.2014.Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens[J].Virus Disease,25(1):114-119.
Morshed R,Hosseini H,Langeroudi A G,et al.2017.Fowl adenoviruses D and E cause inclusion body hepatitis outbreaks in broiler and broiler breeder pullet flocks[J]Avian Diseases,61(2):205-210.
Niu Y J,Sun W,Zhang G H,et al.2016.Hydropericardium syndrome outbreak caused by fowl adenovirus serotype4 in China in 2015[J].The Journal of General Virology97(10):2684-2690.
Pitcovski J,Mualem M,Rei-Koren Z,et al.1998.The complete DNA sequence and genome organization of the avian adenovirus,hemorrhagic enteritis virus[J].Virology,249(2):307-315.
Said A,Wang W,Woldermariam T,et al.2018.Domains of bovine adenovirus-3 protein 22K involved in interacting with viral protein 52K and cellular importinsα-5/α-7[J].Virology,522:209-219.
To K K,Tse H,Chan W M,et al.2014.A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis:Zoonosis associated with virus-bacterium coinfection in birds[J].PLoS Neglected Tropical Diseases,8(12):e3318.
Uchiyama M,Maesawa C,Yashima-Abo A,et al.2005.Consensus JH gene probes with conjugated 3'-minor groove binder for monitoring minimal residual disease in acute lymphoblastic leukemia[J].The Journal of Molecular Diagnostics,7(1):121-126.
Van Eck J H,Davelaar F G,Heuvel-Plesman T A,et al.1976.Dropped egg production,soft shelled and shell-less eggs associated with appearance of precipitins to adenovirus in flocks of laying fowls[J].Avian Pathology,5(4):261-272.
Wang X,Qi X,Yang B,et al.2018.Autophagy benefits the replication of egg drop syndrome virus in duck embryo fibroblasts[J].Frontiers in Microbiology,9:1091
Wu K,Orozco D,Hearing P.2012.The adenovirus L4-22Kprotein is multifunctional and is an integral component of crucial aspects of infection[J].Journal of Virology,86(19):10474-10483.
Zhang X,Zhong Y,Zhou Z,et al.2016.Molecular characterization,phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014[J].Virology,493:12-21.
Ahi Y S,Mittal S K.2016.Components of adenovirus genome packaging[J].Frontiers in Microbiology,7:1503
Biasiotto R,Akusj?rvi G.2015.Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33Kand L4-22K proteins[J].International Journal of Molecular Sciences,16(2):2893-2912.
Bouquet J F,Moreau Y,McFerran J B,et al.1982.Isolation and characterisation of an adenovirus isolated from Muscovy ducks[J].Avian Pathology,11(2):301-307.
Cha S Y,Kang M,Moon O K,et al.2013.Respiratory disease due to current Egg drop syndrome virus in Pekin ducks[J].Veterinary Microbiology,165(3-4):305-311.
Das S,Fearnside K,Sarker S,et al.2017.A novel pathogenic aviadenovirus from red-bellied parrots(Poicephalus rufiventris)unveils deep recombination events among avian host lineages[J].Virology,502:188-197.
Guimet D,Hearing P.2013.The adenovirus L4-22K protein has distinct functions in the posttranscriptional regulation of gene expression and encapsidation of the viral genome[J].Journal of Virology,87(13):7688-7699.
Kaján G L,Stefancsik R,Ursu K,et al.2010.The first complete genome sequence of a non-chicken aviadenovirus,proposed to be turkey adenovirus 1[J].Virus Research,153(2):226-233.
Kang M,Cha S Y,Jang H K.2017.Tropism and infectivity of duck-derived egg drop syndrome virus in chickens[J].PLoS One,12(5):e0177236.
Kutyavin I V,Afonina I A,Mills A,et al.2000.3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures[J].Nucleic Acids Research,28(2):655-661.
Lan S,Kamel W,Punga T,et al.2017.The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding[J].Nucleic Acids Research,45(4):1731-1742.
Li C J,Li H Y,Wang D D,et al.2016.Characterization of fowl adenoviruses isolated between 2007 and 2014 in China[J].Veterinary Microbiology,197():62-67.
Marek A,Kaján G L,Kosiol C,et al.2014.Complete genome sequences of pigeon adenovirus 1 and duck adenovirus2 extend the number of species within the genus Aviadenovirus[J].Virology,462-463:107-114.
Marek A,Ballmann M Z,Kosiol C,et al.2014.Whole-genome sequences of two turkey adenovirus types reveal the existence of two unknown lineages that merit the establishment of novel species within the genus Aviadenovirus[J]The Journal of General Virology,95(1):156-170.
Mittal D,Jindal N,Tiwari A K,et al.2014.Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens[J].Virus Disease,25(1):114-119.
Morshed R,Hosseini H,Langeroudi A G,et al.2017.Fowl adenoviruses D and E cause inclusion body hepatitis outbreaks in broiler and broiler breeder pullet flocks[J]Avian Diseases,61(2):205-210.
Niu Y J,Sun W,Zhang G H,et al.2016.Hydropericardium syndrome outbreak caused by fowl adenovirus serotype4 in China in 2015[J].The Journal of General Virology97(10):2684-2690.
Pitcovski J,Mualem M,Rei-Koren Z,et al.1998.The complete DNA sequence and genome organization of the avian adenovirus,hemorrhagic enteritis virus[J].Virology,249(2):307-315.
Said A,Wang W,Woldermariam T,et al.2018.Domains of bovine adenovirus-3 protein 22K involved in interacting with viral protein 52K and cellular importinsα-5/α-7[J].Virology,522:209-219.
To K K,Tse H,Chan W M,et al.2014.A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis:Zoonosis associated with virus-bacterium coinfection in birds[J].PLoS Neglected Tropical Diseases,8(12):e3318.
Uchiyama M,Maesawa C,Yashima-Abo A,et al.2005.Consensus JH gene probes with conjugated 3'-minor groove binder for monitoring minimal residual disease in acute lymphoblastic leukemia[J].The Journal of Molecular Diagnostics,7(1):121-126.
Van Eck J H,Davelaar F G,Heuvel-Plesman T A,et al.1976.Dropped egg production,soft shelled and shell-less eggs associated with appearance of precipitins to adenovirus in flocks of laying fowls[J].Avian Pathology,5(4):261-272.
Wang X,Qi X,Yang B,et al.2018.Autophagy benefits the replication of egg drop syndrome virus in duck embryo fibroblasts[J].Frontiers in Microbiology,9:1091
Wu K,Orozco D,Hearing P.2012.The adenovirus L4-22Kprotein is multifunctional and is an integral component of crucial aspects of infection[J].Journal of Virology,86(19):10474-10483.
Zhang X,Zhong Y,Zhou Z,et al.2016.Molecular characterization,phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014[J].Virology,493:12-21.