Ⅰ群禽腺病毒血清4型TaqMan探针实时荧光定量PCR检测方法的建立及应用
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摘要
为储备禽腺病毒病疫情防控技术,应用DNAStar软件分析GenBank中Ⅰ群禽腺病毒血清4型(FAdV-4)全基因序列,选取Hexon基因的保守区域设计合成1对PCR引物和1条TaqMan探针,建立FAdV-4 FQ-PCR检测方法,得到相应的标准曲线,并应用该方法对临床病例和人工感染动物进行FAdV-4核酸检测。结果表明,建立的FAdV-4 FQ-PCR方法具有良好的敏感性、特异性、稳定性和临床应用性。
In order to reserve the technology for prevention and control of avian adenovirus disease, the whole gene sequences of groupⅠavian adenovirus serotype 4(FADV-4) were analyzed with the DNAStar software and the conserved sequence of Hexon gene was selected to synthesize a pair of PCR primers and a TaqMan probe.FAdV-4 FQ-PCR detection method and corresponding standard curve were established and used to detect the samples of clinical cases and artificially infected cases.The results showed that the FAdV-4 FQ-PCR method had good sensitivity, specificity, stability and clinical applicability.The establishment of this method will promote the research in rapid detection and pathogenes of Fowl Adenovirus groupⅠSerotype 4.
In order to reserve the technology for prevention and control of avian adenovirus disease, the whole gene sequences of groupⅠavian adenovirus serotype 4(FADV-4) were analyzed with the DNAStar software and the conserved sequence of Hexon gene was selected to synthesize a pair of PCR primers and a TaqMan probe.FAdV-4 FQ-PCR detection method and corresponding standard curve were established and used to detect the samples of clinical cases and artificially infected cases.The results showed that the FAdV-4 FQ-PCR method had good sensitivity, specificity, stability and clinical applicability.The establishment of this method will promote the research in rapid detection and pathogenes of Fowl Adenovirus groupⅠSerotype 4.
引文
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[21] 蔡宇威,刘兰,梁雄燕,等.1例鸡安卡拉病的诊断及病原分离鉴定[J].畜牧与兽医,2016,48(12):89-91.CAI Y W,LIU L,LIANG X Y,et al.Diagnosis and pathogen identification of a case of chicken Ankara disease[J].Animal Husbandry & Veterinary Medicine,2016,48(12):89-91.
[22] 程敏.猪瘟病毒实时定量PCR检测方法的建立及初步应用[J].西北农业学报,2012,21(9):29-33.CHENG M.Establishment and preliminary application of real-time PCR assav for quantitative detection of classical swine fever virus[J].Acta Agriculturae Boreali-occidentalis Sinica,2012,21(9):29-33.
[23] 程振涛,张双翔,王慧,等.绵羊肺炎支原体实时荧光定量PCR检测方法的建立[J].西北农业学报,2013,22(2):7-12.CHENG ZH T,ZHANG SH X,WANG H,et al.Establishment of real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae[J].Acta Agriculturae Boreali-occidentalis Sinica,2013,22(2):7-12.
[24] PAN Q,LIU L,GAO Y,et al.Characterization of a hypervirulent fowl adenovirus 4 with the novel genotype newly prevalent in China and establishment of reproduction infection model of hydropericardium syndrome in chickens[J].Poultry Science,2017,96(6):1581.
[25] TORO H,GONZALEZ C,CERDA L,et al.Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus[J].Avian Diseases,2002,46(3):547-554.
[2] YE J,LIANG G,ZHANG J,et al.Outbreaks of serotype 4 fowl adenovirus with novel genotype,China[J].Emerging Microbes & Infections,2016,5(5):e50.
[3] 张曼,韩飞,高睿,等.Ⅰ群禽腺病毒Hexon蛋白的原核表达及间接ELISA检测方法的建立与应用[J].西北农业学报,2018,27(5):634-640.ZHANG M,HAN F,GAO R,et al.Establishment and application of prokaryotic expression of Hexon protein of fowl adenovirus groupⅠ and indirect ELISA in Escherichia coli [J].Acta Agriculturae Boreali-occidentalis Sinica,2018,27(5):634-640.
[4] CHANRA R,SHUKLA S K,KUMAR M.The hydropericardium syndrome and inclusion body hepatitis in domestic fowl[J].Tropical Animal Health & Production,2000,32(2):99-111.
[5] 马震原.荧光定量PCR检测EDSV方法的建立与应用及NE_4毒株感染特性的研究[D].成都:四川农业大学,2012.MA ZH Y.Development of TaqMan real-time PCR for the detection of EDSVand studies on infectious characteristics of NE4 strain[D].Chengdu:Sichuan Agricultural Uniersity,2012.
[6] 宋庆凯,李晓飞,苗雨润,等.树鼩腺病毒TaqMan实时荧光定量PCR检测方法的建立及初步应用[J].中国比较医学杂志,2018,28(3):73-77.SONG Q K,LI X F,MIAO Y R,et al.Establishment and application of a TaqMan real-time fluorescence quantitative PCR for detection of tree shrew adenovirus(TAV)[J].Chinese Journal of Comparative Medicine,2018,28(3):73-77.
[7] 袁万哲,张姗,陈萍,等.禽腺病毒4型PCR检测方法的建立与应用[J].中国动物检疫,2016,33(5):72-74.YUAN W ZH,ZHANG SH,CHEN P,et al.Development and application of PCR assay for detection on fowl adenovirus 4[J].China Animal Health Inspection,2016,33(5):72-74.
[8] TENG Z,JIN Q,DING P,et al.Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China[J].Virology Journal,2016,13(1):188.
[9] SHAH M S,ASHRAF A,KHAN M I,et al.Molecular cloning,expression and characterization of 100 K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome[J].Biologicals,2016,44(1):19-23.
[10] SHIVACHANDRA S B,SAH R L,SINGH S D,et al.Immunosuppression in broiler chicks fed aflatoxin and inoculated with fowl adenovirus serotype-4 (FAV-4) associated with hydropericardium syndrome[J].Veterinary Research Communications,2003,27(1):39-51.
[11] MCGOLDRICK A,LOWINGS J P,IBATA G,et al.A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan)[J].Journal of Virological Methods,1998,72(2):125.
[12] MEULEMANS G,BOSCHMANS M,BERG T P V D,et al.Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses[J].Avian Pathology,2001,30(6):655-660.
[13] 文艳玲,谢芝勋,王莹,等.Ⅰ群禽腺病毒SYBR GreenⅠ荧光PCR检测方法的建立[J].中国兽医科学,2008,38(9):753-756.WEN Y L,XIE ZH X,WANG Y,et al.Development of a SYBR Green Ⅰ real-time PCR assay for the detection of aviadenovirus group I [J].Chinese Veterinary Science,2008,38(9):753-756.
[14] SHAH M S,ASHRAF A,KHAN M I,et al.Fowl adenovirus:history,emergence,biology and development of a vaccine against hydropericardium syndrome[J].Archives of Virology,2017,162(7):1833.
[15] 唐熠,谢芝勋,熊文婕,等.Ⅰ群禽腺病毒环介导等温扩增检测方法的建立[J].中国兽医科学,2010(7):713-716.TANG Y,XIE ZH X,XIONG W J,et al.Development of a loop-mediated isothermal amplification assay for detection of aviadenovirus group Ⅰ[J].Chinese Veterinary Science,2010(7):713-716.
[16] 陈天骜.血清4型禽腺病毒GX2013毒株的生物学特性鉴定和检测方法的建立[D].江苏扬州:扬州大学,2016.CHEN T A.Identification of biological characteristics of fowladenovirus serotype 4 (FAdV-4) GX2013 strain and establishment of detection methods for FAdV C[D].Yangzhou Jiangsu:Yangzhou University,2016.
[17] 谢芝勋,MazharI Khan.应用PCR检测禽腺病毒[J].中国兽医学报,2000,20(4):21-23.XIE ZH X,MAZHAR K.Detection of avian adenoviruses by polymerase Chain reaction[J].Chinese Journal of Veterinary Science,2000,20(4):21-23.
[18] VANDESOMPELE J,DE P A,SPELEMAN F.Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR greenⅠ real-time RT-PCR[J].Analytical Biochemistry,2002,303(1):95-98.
[19] BROWNIE J,SHAWCROSS S,THEAKER J,et al.The elimination of primer-dimer accumulation in PCR[J].Nucleic Acids Research,1997,25(16):3235-3241.
[20] WANG J,WANG J,CHEN P,et al.Development of a TaqMan-based real-time PCR assay for rapid and specific detection of fowl aviadenovirus serotype 4[J].Avian Pathology,2017,46(3):338-343.
[21] 蔡宇威,刘兰,梁雄燕,等.1例鸡安卡拉病的诊断及病原分离鉴定[J].畜牧与兽医,2016,48(12):89-91.CAI Y W,LIU L,LIANG X Y,et al.Diagnosis and pathogen identification of a case of chicken Ankara disease[J].Animal Husbandry & Veterinary Medicine,2016,48(12):89-91.
[22] 程敏.猪瘟病毒实时定量PCR检测方法的建立及初步应用[J].西北农业学报,2012,21(9):29-33.CHENG M.Establishment and preliminary application of real-time PCR assav for quantitative detection of classical swine fever virus[J].Acta Agriculturae Boreali-occidentalis Sinica,2012,21(9):29-33.
[23] 程振涛,张双翔,王慧,等.绵羊肺炎支原体实时荧光定量PCR检测方法的建立[J].西北农业学报,2013,22(2):7-12.CHENG ZH T,ZHANG SH X,WANG H,et al.Establishment of real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae[J].Acta Agriculturae Boreali-occidentalis Sinica,2013,22(2):7-12.
[24] PAN Q,LIU L,GAO Y,et al.Characterization of a hypervirulent fowl adenovirus 4 with the novel genotype newly prevalent in China and establishment of reproduction infection model of hydropericardium syndrome in chickens[J].Poultry Science,2017,96(6):1581.
[25] TORO H,GONZALEZ C,CERDA L,et al.Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus[J].Avian Diseases,2002,46(3):547-554.