蜜蜂残翅病毒两步法荧光定量PCR 检测方法的建立

doi:10.15933/j.cnki.1004-3268.2022.03.018

摘要/Abstract

摘要： 为了建立一种灵敏度更高的蜜蜂残翅病毒两步法荧光定量PCR检测方法，根据残翅病毒保守基因片段设计引物，采取反转录过程与荧光定量PCR过程分步进行的方式，建立基于SYBR染料法检测残翅病毒的两步法实时荧光定量PCR检测方法，并对其特异性、灵敏性、稳定性和可行性进行验证。结果显示，建立的两步法荧光定量PCR检测方法采用的引物特异性良好，在6.58×101~6.58×108拷贝/μL质粒标准品间具有良好线性关系，R2为0.999 7，扩增效率为100.8%。该方法的灵敏度为6.58拷贝/μL，与蜜蜂黑蜂王台病毒和以色列急性麻痹病毒不发生交叉反应，组内变异系数为0.20%~0.66%，组间变异系数为0.08%~0.98%，对蜜蜂样品残翅病毒的阳性检出率明显高于普通PCR，具有良好的特异性、重复性和实用性。综上，成功建立了一种灵敏度高、适用性好的残翅病毒两步法实时荧光定量PCR检测方法，为我国蜜蜂残翅病的流行病学调查、疫情监测和预警机制的构建提供新的技术支持。

关键词: 西方蜜蜂, 东方蜜蜂, 残翅病毒, 两步法荧光定量PCR, SYBR染料法, 灵敏度

Abstract: In order to establish a more sensitive two⁃step fluorescence quantitative PCR detection method for deformed wing virus in honeybee，primers were designed according to the conservative gene fragment of deformed wing virus，and a two⁃step real⁃time fluorescence quantitative PCR detection method based on SYBR dye was established by conducting reverse transcription process and fluorescence quantitative PCR step by step，and its specificity，sensitivity，stability and feasibility were verified.The results indicated that the primer specificity of the two⁃step real⁃time fluorescence quantitative PCR method established in this study was good，and there was a good linear relationship in 6.58×10¹—6.58×10⁸copies/μL of plasmid standard.R² was 0.999 7 and the amplification efficiency was 100.8%.The sensitivity of the method was 6.58 copies/μL，and there were no cross⁃reactions between deformed wing virus and black queen cell virus，Israeli acute paralysis virus.The coefficient of variation was 0.20%—0.66% within the group and 0.08%—0.98% between the groups.The deformed wing virus infection rate of honeybee samples detected by this method was higher than that by ordinary PCR.The method showed good specificity，repeatability and practicability.In conclusion，a two⁃step real⁃time quantitative PCR method with higher sensitivity and better applicability was successfully established，which provides new technical support for epidemiological investigation，epidemic surveillance and early warning mechanism construction of bee deformed wing virus disease in China.

Key words: Apis mellifera, Apis cerena, Deformed wing virus, Two?step fluorescence quantitative PCR, SYBR dye method, Sensitivity

中图分类号:

S895.9

王红坤, 王艺桦, 周丹银, 董坤, 张炫. 蜜蜂残翅病毒两步法荧光定量PCR 检测方法的建立[J]. 河南农业科学, 2022, 51(3): 154-161.

WANG Hongkun, WANG Yihua, ZHOU Danyin, DONG Kun, ZHANG Xuan. Establishment of Two⁃step Fluorescence Quantitative PCR Method for Detection of Deformed Wing Virus in Honeybee[J]. Journal of Henan Agricultural Sciences, 2022, 51(3): 154-161.

参考文献

［1］王红坤，王艺桦，周丹银，等.蜜蜂残翅病研究进展［J］.蜜蜂杂志，2021，41（10）：9⁃14.

WANG H K，WANG Y H，ZHOU D Y，et al.Review of Deformed wing virus research in the recent decade［J］.Journal of Bee，2021，41（10）：9⁃14.

［2］REMNANT E J，SHI M，BUCHMANN G，et al.A diverse range of novel RNA viruses in geographically distinct honey bee populations［J］.Journal of Virology，2017，91（16）：e00158⁃17.
［3］AMIRI E，KRYGER P，MEIXNER M D，et al.Quantitative patterns of vertical transmission of deformed wing virus in honey bees［J］.PLoS One，2018，13（3）：e0195283⁃12.
［4］ZHANG X，ZHOU D Y，ZHAO W Z，et al.The correlation between the parasitism density of Varroa destructor and deform wing virus replication dynamic［J］.Journal of Yunnan Agricultural University，2014，29（2）：192⁃197.
［5］GROZINGER C M，FLENNIKEN M L.Bee viruses：Ecology，pathogenicity，and impacts［J］.Annual Review of Entomology，2019，64（1）：205⁃226.
［6］ZHANG X，HE S Y，EVANS J D，et al.New evidence that deformed wing virus and black queen cell virus are multihost pathogens ［J］.Journal of Invertebrate Pathology，2012，109（1）：156⁃159.
［7］史红霞，刘永梅，党晓群.蜜蜂RNA病毒在传粉昆虫中的分布及其病害诊断防治方法［J］.河南农业科学，2018，47（1）：1⁃6.
SHI H X，LIU Y M，DANG X Q.The Distribution，diagnosis and prevention methods of honeybee RNA viruses in insect pollinators ［J］.Journal of Henan Agricultural Sciences，2018，47（1）：1⁃6.
［8］DALVISE P，SEEBURGER V，GIHRING K，et al.Seasonal dynamics and co⁃occurrence patterns of honey bee pathogens revealed by high⁃throughput RT⁃qPCR analysis［J］.Ecology and Evolution，2019，9（18）：10241⁃10252.
［9］张炫，陈彦平，和绍禹.蜜蜂病毒学研究进展［J］.应用昆虫学报，2012，49（5）：1095⁃1116.
ZHANG X，CHEN Y P，HE S Y.A review of bee virology progress［J］.Chinese Journal of Applied Entomology，2012，49（5）：1095⁃1116.
［10］王琛，马跃宇，黄思超，等.蜜蜂残翼病毒TaqMan-MGB探针荧光定量RT-PCR检测方法的建立［J］.病毒学报，2021，37（3）：686⁃694.
WANG C，MA Y Y，HUANG S C，et al.Development of a quantitative PCR method using a TaqMan⁃MGB fluorescent probe for detection of the deformed wing virus［J］.Chinese Journal of Virology，2021，37（3）：686⁃694.
［11］SIMEUNOVIC P，STEVANOVIC J，VIDANOVIC D，et al.A survey of deformed wing virus and acute bee paralysis virus in honey bee colonies from Serbia using real⁃time RT⁃PCR［J］.Acta Veterinaria⁃Beograd，2014，64（1）：81⁃92.
［12］SCHURR F，TISON A，MILITANO L，et al.Validation of quantitative real⁃time RT⁃PCR assays for the detection of six honeybee viruses［J］.Journal of Virological Methods，2019，270（8）：70⁃78.
［13］张晟春.两种荧光定量PCR法检测乳腺癌组织中miR-21的应用分析［D］.遵义：遵义医学院，2012.
ZHANG S C.Comparison of microRNA⁃21 in breast cancer tissue detection results with two fluorescent quantitative PCR methods［D］.Zunyi：Zunyi Medical University，2012.
［14］DING G L，SHI W.Investigation of seven viruses in Apis cerana and Apis mellifera of China［J］.Acta Veterinaria et Zootechnica Sinica，2015，46（10）：1822⁃1828.
［15］王蕾，马啸天，和绍禹.蜜蜂畸翅病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立［J］.蜜蜂杂志，2012，32（4）：3⁃6.
WANG L，MA X T，HE S Y.A real time quantitative RT⁃PCR assay for the detection of deformed wing virus in honeybee［J］.Journal of Bee，2012，32（4）：3⁃6.
［16］DUBOIS E，DARDOURI M，SCHURR F，et al.Outcomes of honeybee pupae inoculated with deformed wing virus genotypes A and B［J］.Apidologie，2020，51（1）：18⁃34.
［17］FELICIOLI A，FORZAN M，SAGONA S，et al.Effect of oral administration of 1，3⁃1，6 β⁃glucans in DWV naturally infected newly emerged bees（Apis mellifera L.）［J］.Veterinary Sciences，2020，7（2）：52⁃62.
［18］ABD⁃EL⁃SAMIE E M，ADHAM F K，EL⁃MOHANDES S，et al.First detection of deformed wing and kakugo viruses in honeybees（Apis mellifera L.）in Egypt by real time⁃polymerase chain reaction（RT⁃PCR）［J］.African Journal of Biotechnology，2017，16（14）：738⁃748.
［19］贾慧茹，周婷，高夫超.一步法RT-PCR和两步法RTPCR对蜜蜂病毒诊断研究的比较［J］.应用昆虫学报，2015，52（2）：351⁃355.
JIA H R，ZHOU T，GAO F C.Research on the detection of bee viruses by one step and two step RT⁃PCR［J］.Chinese Journal of Applied Entomology，2015，52（2）：351⁃355.
［20］韦信贤，陈孝宇，贾鹏，等.虾虹彩病毒TaqMan-MGB探针荧光定量PCR检测方法的建立［J］.南方农业学报，2020，51（2）：404⁃411.
WEI X X，CHEN X Y，JIA P，et al.Development of TaqMan⁃MGB probe fluorescence quantitative PCR for detection of shrimp iridescent virus［J］.Journal of Southern Agriculture，2020，51（2）：404⁃411.
［21］汪伟，杜倩，韩知晓，等.痘苗病毒SYBR Green Ⅰ荧光定量PCR检测方法的建立与应用［J］.中国兽医科学，2020，50（4）：430⁃436.
WANG W，DU Q，HAN Z X.Establishment and application of SYBR Green Ⅰ real⁃time PCR method for detection of vaccinia virus［J］.Chinese Veterinary Science，2020，50（4）：430⁃436.
［22］陈少杰，刘立辉，吴志勇，等.猪星状病毒4型SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立［J］.畜牧兽医学报，2020，51（2）：404⁃408.
CHEN S J，LIU L H，WU Z Y，et al.Establishment and application of a SYBR Green Ⅰ real⁃time PCR technique for the detection of porcine astrovirus 4［J］.Chinese Journal of Animal and Veterinary Sciences，2020，51（2）：404⁃408.
［23］贾云飞，赵福杰，朱静静，等.非洲猪瘟病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立［J］.河南农业大学学报，2020，54（1）：69⁃73，80.
JIA Y F，ZHAO F J，ZHU J J，et al.Development of a SYBR Green I based real⁃time quantitative PCR method
for the detection of African swine fever virus［J］.Journal of Henan Agricultural University，2020，54（1）：69⁃73，
80.
［24］张体银，黄嫦娇，田国宁，等.检测蜜蜂急性麻痹病毒TaqMan实时荧光RT-PCR方法的建立和初步应用［J］.微生物学通报，2021，48（12）：5001⁃5007.
ZHANG T Y，HUANG C J，TIAN G N，et al.Establishment and preliminary application of a real⁃time RT⁃PCR assay with TaqMan probe for detecting acute bee paralysis virus［J］.Microbiology China，2021，48（12）：5001⁃5007.
［25］KOZIY R V，WOOD S C，KOZII I V，et al.Deformed wing virus infection in honey bees（Apis mellifera L.）［J］.Veterinary Pathology，2019，56（4）：636⁃641.
［26］MORDECAI G J，WILFERT L，MARTIN S J，et al.Diversity in a honey bee pathogen：First report of a third master variant of the deformed wing virus quasispecies［J］.The ISME Journal，2016，10（5）：1264⁃1273.
［27］MARTIN S J，BRETTELL L E.Deformed wing virus in honeybees and other insects［J］.Annual Review of Virology，2019，6（1）：49⁃69.
［28］ALER S A，BURNHAM P A，BRODY A K.Flowers as viral hot spots：Honey bees（Apis mellifera）unevenly deposit viruses across plant species［J］.PLoS One，2019，14（9）：e0221800⁃16.
［29］CHEN Y P，PETTIS J S，COLLINS A，et al.Prevalence and transmission of honeybee viruses［J］.Applied and Environmental Microbiology，2006，72（1）：606⁃611.
［30］周新宇，陈鑫鑫，乔松林，等.猪繁殖与呼吸综合征病毒类NADC30毒株与JXA1-R疫苗株荧光定量PCR鉴别检测方法的建立及应用［J］.河南农业科学，2021，50（1）：142⁃151.
ZHOU X Y，CHEN X X，QIAN S L，et al.Establishment and application of real⁃time PCR for differentiation of porcine reproductive and respiratory syndrome virus strains NADC30⁃like and JXA1⁃R［J］.Journal of Henan Agricultural Sciences，2021，50（1）：142⁃151.
［31］WU Y，DONG X，KADOWAKI T.Characterization of the copy number and variants of deformed wing virus（DWV）in the pairs of honey bee pupa and infesting Varroa destructor or Tropilaelaps mercedesae［J］.Frontiers in Microbiology，2017，22（8）：1558.
［32］DIAO Q，YANG D，ZHAO H，et al.Prevalence and population genetics of the emerging honey bee pathogen DWV in Chinese apiculture［J］.Scientific Reports，2019，9（1）：1⁃10.

[1]	王文海;曹殿立;. 影子价格与灵敏度分析在施肥方案调整中的应用[J]. 河南农业科学, 2009, 38(1): 59-63.
[2]	陈建军;曹香林;李学梅;刘崇怀;古勤生. PAS-ELISA检测GFLV和GLRaV-3技术的研究[J]. 河南农业科学, 2007, 36(2): 50-53.
[3]	陈建军 ;李培睿 ;杨向英 ;曹香林 ;刘崇怀. DAS-ELISA和PAS-ELISA检测GFLV的技术研究[J]. 河南农业科学, 2004, 33(11): 48-51.
[4]	吴坤,徐淑霞,陈红歌,张跃灵. 土壤中五氯酚的测定及其生物降解研究[J]. 河南农业科学, 2003, 32(5): 30-33.