应用等温环介导扩增方法检测蜜蜂残翅病毒的研究
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摘要
本文的目的在于建立用于临床检测残翅病病毒(Deformed wing virus,DWV)的等温环介导扩增技术(Loopmediated isothermal amplification,LAMP),为该疾病的预防和控制提供理论依据。在DWV基因保守序列设计4条引物,探究LAMP扩增的最优条件,并与常规的PCR(polymerase chain reaction)检测方法进行比较。建立的LAMP方法检测下限为0.89 pg,灵敏度比PCR高100倍而且特异性好。临床检测显示建立的LAMP方法可行、准确、方便、灵敏。针对DWV的LAMP建立的检测方法为养蜂生产第一线检测和预防DWV提供了技术支持,有一定的应用价值。
The objective of this study is to establish a simple,fast and accurate method to deformed wing virus( DWV) in clinical by using loop-mediated isothermal amplification( LAMP),which provides an experimental proof for controlling the disease. Four primers were designed based on six conserved regions of gene sequence on DWV,and were used for exploring the optimal LAMP amplification conditions. And the LAMP amplification result was compared conventional PCR( polymerase chain reaction) method. The established LAMP could detect as low as 0. 89 pg DNA,which was 100 times sensitive than PCR method.Clinical result showed LAMP could be used to detect DWV whatever in Italian bee( Apis mellifera ligustica)or in Chinese bee( A. cerana cerana),and the real positive detected ratio was 20% higher than PCR tested. The established LAMP was practical valuable for detecting and controlling the DWV in honeybee keeping.
The objective of this study is to establish a simple,fast and accurate method to deformed wing virus( DWV) in clinical by using loop-mediated isothermal amplification( LAMP),which provides an experimental proof for controlling the disease. Four primers were designed based on six conserved regions of gene sequence on DWV,and were used for exploring the optimal LAMP amplification conditions. And the LAMP amplification result was compared conventional PCR( polymerase chain reaction) method. The established LAMP could detect as low as 0. 89 pg DNA,which was 100 times sensitive than PCR method.Clinical result showed LAMP could be used to detect DWV whatever in Italian bee( Apis mellifera ligustica)or in Chinese bee( A. cerana cerana),and the real positive detected ratio was 20% higher than PCR tested. The established LAMP was practical valuable for detecting and controlling the DWV in honeybee keeping.
引文
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Dinh DT,Le MT,Vuong CD,et al.An updated loop-mediated isothermal amplification method for rapid diagnosis of H5N1 avian influenza viruses[J].Tropical Medicine Health,2011,39(1):3-7.
Ellis JD,Munn PA.The wordwide health status of honey bees[J].Bee World,2005,86(4):88-101.
Gaetana L,Miranda JR,MaRIA BB,et al.Molecular and biological characterization of deformed wing virus of honey bees(Apis mellifera)[J].Journal of Virology,2006,80(10):4998-5009.
Jia HR,Liu JZ,Wang X,et al.Occurrence and prevalence of six bee viruses in Beijing[J].Chinese Journal of Applied Entomology,2014,51(3):772-780.[贾慧茹,刘进祖,王星,等.北京地区六种蜜蜂病毒病的流行病学研究[J].应用昆虫学报,2014,51(3):772-780]
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Miranda JR,Genersch E.Deformed wing virus[J].Journal of Invertebrate Pathology,2010,103(1):48-61.
Neumann P,Carreck NL.Honey bee colony losses[J].Journal of Apicultural Research,2010,49(1):1-6.
Tentcheva D,Gauthier L,Jouve S,et al.Polymerase chain reaction detection of deformed wing virus(DWV)in Apis mellifera and Varroa destructor[J].Apidologie,2004,35(4):431-439.
Tang FF,Yang WK,Shao YL,et al.Prompt diagnosis of the Bombyx mori nucleopolyhedrosis virus by loop-mediated isothermal amplification[J].Chinese Journal of Applied Entomology,2013,50(4):1013-1019.[唐芬芬,杨伟克,邵榆岚,等.应用环介导等温扩增技术(LAMP)快速检测家蚕核型多角体病毒的研究[J].应用昆虫学报,2013,50(4):1013-1019]
Yuan DW,Liu JS,Guo DC,et al.Application of a loop-mediated isothermal amplification method in detection of RHDV[J].Chinese Veterinary Science,2013,43(4):390-395.[原冬伟,刘家森,郭东春,等.环介导等温扩增技术兔出血症病毒检测中的应用[J].中国兽医科学,2013,43(4):390-395]
Chen YP,Siede R.Honey bee viruses[J].Advances in Virus Research,2007,70(7):33-80.
Dinh DT,Le MT,Vuong CD,et al.An updated loop-mediated isothermal amplification method for rapid diagnosis of H5N1 avian influenza viruses[J].Tropical Medicine Health,2011,39(1):3-7.
Ellis JD,Munn PA.The wordwide health status of honey bees[J].Bee World,2005,86(4):88-101.
Gaetana L,Miranda JR,MaRIA BB,et al.Molecular and biological characterization of deformed wing virus of honey bees(Apis mellifera)[J].Journal of Virology,2006,80(10):4998-5009.
Jia HR,Liu JZ,Wang X,et al.Occurrence and prevalence of six bee viruses in Beijing[J].Chinese Journal of Applied Entomology,2014,51(3):772-780.[贾慧茹,刘进祖,王星,等.北京地区六种蜜蜂病毒病的流行病学研究[J].应用昆虫学报,2014,51(3):772-780]
Liang Q,Chen DF.Bee Protection[M].Beijing:Chinese Agricultural Press,2009:216.[梁勤,陈大福.蜜蜂保护学[M].北京:中国农业出版社,2009:216]
Martin SJ,Ball BV,Carreck NL,et al.Prevalence and persistence of deformed wing virus(DWV)in untreated or acaricide-treated Varroa destructor infested honey bees(Apis mellifera)colonies[J].Journal of Apicultural Research,2010,49(1):72-79.
Miranda JR,Genersch E.Deformed wing virus[J].Journal of Invertebrate Pathology,2010,103(1):48-61.
Neumann P,Carreck NL.Honey bee colony losses[J].Journal of Apicultural Research,2010,49(1):1-6.
Tentcheva D,Gauthier L,Jouve S,et al.Polymerase chain reaction detection of deformed wing virus(DWV)in Apis mellifera and Varroa destructor[J].Apidologie,2004,35(4):431-439.
Tang FF,Yang WK,Shao YL,et al.Prompt diagnosis of the Bombyx mori nucleopolyhedrosis virus by loop-mediated isothermal amplification[J].Chinese Journal of Applied Entomology,2013,50(4):1013-1019.[唐芬芬,杨伟克,邵榆岚,等.应用环介导等温扩增技术(LAMP)快速检测家蚕核型多角体病毒的研究[J].应用昆虫学报,2013,50(4):1013-1019]
Yuan DW,Liu JS,Guo DC,et al.Application of a loop-mediated isothermal amplification method in detection of RHDV[J].Chinese Veterinary Science,2013,43(4):390-395.[原冬伟,刘家森,郭东春,等.环介导等温扩增技术兔出血症病毒检测中的应用[J].中国兽医科学,2013,43(4):390-395]