毛皮兽阿留申病毒可视化交叉等温扩增检测方法的建立及初步应用
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摘要
为建立快速、特异、灵敏的毛皮兽阿留申病毒可视化交叉引物等温扩增检测方法(CPA),以阿留申病毒属内病毒序列为参考,在VP2序列高变区保守区段设计合成4条特异引物。通过优化CPA反应条件,建立了毛皮兽阿留申病毒可视化交叉引物等温扩增检测方法,并进行了特异性、敏感性试验以及对临床样品的检测。结果显示,该方法可扩增水貂阿留申病毒(AMDV)和狐貉阿留申病毒(RFAV),对犬科和水貂组织以及常见病毒无特异性反应;对AMDV、RFAV可视化检测下限分别为5.38×10~2copies/μL、5. 93×10~2copies/μL;对83份临床样品的检测结果显示,其中有16份PCR检测为阴性,CPA、qPCR检测为阳性的样品,表明CPA检测方法优于传统PCR。本试验建立了一种高敏感性、特异性检测毛皮兽阿留申病毒的方法,通过添加荧光染料实现了可视化检测,它更加适合基层兽医站、养殖场进行快速核酸检测。
To establish a rapid,specific and sensitive method for the detection of fur animal amdoparvovirus visual by using cross-priming amplification(CPA),four primers was designed and synthesized.They were located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in Gen Bank.The reaction conditions were optimized,the specificity and sensitivity were determined,and clinical samples were detected.In result,the method may be used to amplify AMDV and RFAV with no cross reaction with canine and mink tissue and common viruses.The minimum concentrations of visual detection for AMDV and RFAV were 5.38×10~2 copies/μ L and 5.93×10~2 copies/μL,respectively.Out of 83 clinical specimens,16 samples are PCR negative,but both q PCR and CPA positive,indicating that the sensitivity of CPA is superior to the traditional PCR.In conclusion,the method has a high sensitivity and specific detection of fur animal amdoparvovirus,which is visually detected by fluorochrome.It can provide a widely applicable technique for rapid quarantine,which is more suitable,especially in resource limited laboratories and farms as well as in field conditions.
To establish a rapid,specific and sensitive method for the detection of fur animal amdoparvovirus visual by using cross-priming amplification(CPA),four primers was designed and synthesized.They were located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in Gen Bank.The reaction conditions were optimized,the specificity and sensitivity were determined,and clinical samples were detected.In result,the method may be used to amplify AMDV and RFAV with no cross reaction with canine and mink tissue and common viruses.The minimum concentrations of visual detection for AMDV and RFAV were 5.38×10~2 copies/μ L and 5.93×10~2 copies/μL,respectively.Out of 83 clinical specimens,16 samples are PCR negative,but both q PCR and CPA positive,indicating that the sensitivity of CPA is superior to the traditional PCR.In conclusion,the method has a high sensitivity and specific detection of fur animal amdoparvovirus,which is visually detected by fluorochrome.It can provide a widely applicable technique for rapid quarantine,which is more suitable,especially in resource limited laboratories and farms as well as in field conditions.
引文
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[17]KOWALCZYK M,JAKUBCZAK A,HORECKA B,et al.A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection[J].Virus Genes,2018,54(4):561-569.
[18]ZHANG Z,WANG B,HU S,et al.Detection of Aleutian disease virus by loop-mediated isothermal amplification[J].Virus Disease,2015,26(3):203-206.
[19]田国宁,张金玲,李凯,等.水貂阿留申病毒环介导等温扩增(LAMP)检测方法的建立[J].中国动物检疫,2015,32(6):79-82.TIAN Guo-ning,ZHANG Jin-ling,LI Kai,et al.Development of loop-mediated isothermal amplification(LAMP)assay for detection of Aleutian disease virus(ADV)[J].China Animal Health Inspection,2015,32(6):79-82.(in Chinese)
[20]张攀松.基于恒温扩增的RNA检测方法的研究[D].青岛:青岛科技大学,2017.ZHANG Pan-song.RNA based isothermal amplification detection method[D].Qingdao:Qingdao University of Science,2017.(in Chinese)
[21]FARID A H,RUPASINGHE P P.A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink[J].Canadian Journal of Microbiology,2017,63(4):341-349.
[2]CANUTI M,DOYLE H E,BRITTON A P,et al.Full genetic characterization and epidemiology of a novel amdoparvovirus in striped skunk(Mephitis mephitis)[J].Emerging Microbes&Infections,2017,6(5):e30.
[3]LI L,PESAVENTO P A,WOODS L,et al.Novel amdovirus in gray foxes[J].Emerging Infectious Diseases,2011,17(10):1876-1878.
[4]BODEWES R,RUIZ-GONZALEZ A,SCHAPENDONK C M,et al.Viral metagenomic analysis of feces of wild small carnivores[J].Virology Journal,2014,11:89.
[5]SHAO X Q,WEN Y J,BA H X,et al.Novel amdoparvovirus infecting farmed raccoon dogs and arctic foxes[J].Emerging Infectious Diseases,2014,20(12):2085-2088.
[6]ALEX C E,KUBISKI S V,LI L,et al.Amdoparvovirus infection in red pandas(Ailurus fulgens)[J].Veterinary Pathology,2018,55(4):552-561.
[7]邵西群,章秀婷,于淼,等.貉狐源阿留申病病毒的感染检测与基因特征[J].中国兽医科学,2015,45(6):589-595.SHAO Xi-qun,ZHANG Xiu-ting,YU Miao,et al.Detection of raccoon dog and fox amdoparvovirus infection and viral genetic characteristics[J].Chinese Veterinary Science,2015,45(6):589-595.(in Chinese)
[8]XU G,HU L,ZHONG H,et al.Cross priming amplification:mechanism and optimization for isothermal DNAamplification[J].Scientific Reports,2012,2:246.
[9]FANG R,LI X,HU L,et al.Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimens[J].Journal of Clinical Microbiology,2009,47(3):845-847.
[10]NICZYPORUK J S,WOZNIAKOWSKI G,SAMOREK-SALAMONOW-ICZ E.Application of cross-priming amplification(CPA)for detection of fowl adenovirus(FAdV)strains[J].Archives of Virology,2015,160(4):1005-1013.
[11]霍亚云.四种重要植物病原物恒温扩增检测技术的建立[D].北京:中国农业科学院,2018.HUO Ya-yun.Development of detection methods for four important plant pathogens on the base of isothermal amplification[D].Beijing:Chinese Academy of Agricultural Sciences,2018.(in Chinese)
[12]高琳,王淞,刘国红,等.等温扩增技术在食品中沙门氏菌检测中的应用[J].食品安全质量检测学报,2017,8(1):210-215.GAO Lin,WANG Song,LIU Guo-hong,et al.Application of isothermal amplification technique in the detection of Salmonella in food[J].Journal of Food Safety and Quality,2017,8(1):210-215.(in Chinese)
[13]BLOOM M E,BEST S M,HAYES S F,et al.Identification of aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection,virus neutralization,and immune complex formation[J].Journal of Virology,2001,75(22):11116-11127.
[14]DWORAJK L J,WOLFINBARGER J B,BlOOM M E.Aleutian mink disease parvovirus infection of K562 cells is antibody-dependent and is mediated via an Fc(gamma)RⅡreceptor[J].Archives of Virology,1997,142(2):363-373.
[15]刘艺,冷雪,时坤,等.水貂阿留申病毒检测方法研究进展[J].动物医学进展,2018,39(10):74-77.LIU Yi,LENG Xue,SHI Kun,et al.Progress on detection method of Aleutian disease virus[J].Progress in Veterinary Medicine,2018,39(10):74-77.(in Chinese)
[16]QIU J,CHENG F,BURGER L R,et al.The transcription profile of Aleutian mink disease virus in CRFKcells is generated by alternative processing of pre-mRNAs produced from a single promoter[J].Journal of Virology,2006,80(2):654-662.
[17]KOWALCZYK M,JAKUBCZAK A,HORECKA B,et al.A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection[J].Virus Genes,2018,54(4):561-569.
[18]ZHANG Z,WANG B,HU S,et al.Detection of Aleutian disease virus by loop-mediated isothermal amplification[J].Virus Disease,2015,26(3):203-206.
[19]田国宁,张金玲,李凯,等.水貂阿留申病毒环介导等温扩增(LAMP)检测方法的建立[J].中国动物检疫,2015,32(6):79-82.TIAN Guo-ning,ZHANG Jin-ling,LI Kai,et al.Development of loop-mediated isothermal amplification(LAMP)assay for detection of Aleutian disease virus(ADV)[J].China Animal Health Inspection,2015,32(6):79-82.(in Chinese)
[20]张攀松.基于恒温扩增的RNA检测方法的研究[D].青岛:青岛科技大学,2017.ZHANG Pan-song.RNA based isothermal amplification detection method[D].Qingdao:Qingdao University of Science,2017.(in Chinese)
[21]FARID A H,RUPASINGHE P P.A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink[J].Canadian Journal of Microbiology,2017,63(4):341-349.