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浙江大学学报(农业与生命科学版)  2019, Vol. 45 Issue (3): 385-390    DOI: 10.3785/j.issn.1008-9209.2018.07.181
动物科学与动物医学     
浙江省淳安县中华蜜蜂蜂房球菌聚合酶链式反应检测方法的建立与应用
郭蕊1(),董捷1,郭海坤2,李君竹3,王德前1()
1. 浙江省农业科学院畜牧兽医研究所,杭州 310021
2. 浙江省农业科学院农产品质量标准研究所,杭州 310021
3. 浙江省淳安千岛湖好清蜜蜜蜂专业合作社,杭州 311701
Establishment and application of a polymerase chain reaction method for detection of Melissococcus pluton in honeybee (Apis cerana cerana) in Chunan County of Zhejiang Province
Rui GUO1(),Jie DONG1,Haikun GUO2,Junzhu LI3,Deqian WANG1()
1. Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. Institute of Quality and Standard for Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Hao Qing-mi Beekeeping Professional Cooperative of Qiandao Lake of Zhejiang, Hangzhou 311701, China
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摘要:

通过建立蜂房球菌(Melissococcus pluton)的聚合酶链式反应(polymerase chain reaction, PCR)检测方法,对浙江省淳安县主要中华蜜蜂(Apis cerana cerana)饲养区进行欧洲幼虫腐臭病的流行病学调查。结果显示,新建立的蜂房球菌PCR检测方法特异性强,灵敏度高,并具有良好的重复性和准确性。流行病学调查表明,在淳安县17个乡镇的298群蜜蜂样品中,共有11个乡镇检出蜂房球菌,检出率为25.2%,其中5个乡镇的阳性检出率≥40%。总之,本研究建立了一个新的蜂房球菌PCR检测方法,明确了淳安县中华蜜蜂对欧洲幼虫腐臭病的感染情况,为合理制定该病害的防治措施提供了科学依据。

关键词: 中华蜜蜂蜂房球菌欧洲幼虫腐臭病聚合酶链式反应淳安县流行病学    
Abstract:

A polymerase chain reaction (PCR) method was developed to detect Melissococcus pluton and an epidemiological investigation of European foulbrood (EFB) was done in the main bee (Apis cerana cerana) breeding area of Chun’an County, Zhejiang Province. Results indicated that the newly developed method was specific, sensitive, repeatable and accuracy. The epidemiological survey results showed that a total of 11 townships were infected by M. pluton among the 298 bee samples in 17 townships of Chun’an County. The total infection rate was 25.2%, of which EFB infection rate of five townships was ≥40%. In a word, the newly established method for PCR detection makes the infection status of EFB clear in Chun’an County, and it lays a scientific basis for formulating rationally prevention measures of EFB.

Key words: Apis cerana cerana    Melissococcus pluton    European foulbrood    polymerase chain reaction    Chun’an County    epidemiology
收稿日期: 2018-07-18 出版日期: 2019-06-25
CLC:  S 895.132  
基金资助: 浙江省农业科学院地方科技合作项目(CA20170001)
通讯作者: 王德前     E-mail: 754894816@qq.com;wangdeqian2002@sohu.com
作者简介: 郭蕊(https://orcid.org/0000-0002-4800-9819),E-mail:754894816@qq.com
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引用本文:

郭蕊,董捷,郭海坤,李君竹,王德前. 浙江省淳安县中华蜜蜂蜂房球菌聚合酶链式反应检测方法的建立与应用[J]. 浙江大学学报(农业与生命科学版), 2019, 45(3): 385-390.

Rui GUO,Jie DONG,Haikun GUO,Junzhu LI,Deqian WANG. Establishment and application of a polymerase chain reaction method for detection of Melissococcus pluton in honeybee (Apis cerana cerana) in Chunan County of Zhejiang Province. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 385-390.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.07.181        http://www.zjujournals.com/agr/CN/Y2019/V45/I3/385

图1   PCR的退火温度优化
图2  重组质粒的双酶切鉴定结果
图3  敏感性试验结果
图4   PCR检测方法的特异性试验结果
图5  部分样品蜂房球菌的PCR检测结果

序号

No.

乡镇

Town

蜂群(群)

Colony

阳性数(群)

Positive number

蜂房球菌检出率

Positive rate/%

合计 Total 298 75 25.2
1 威坪镇 10 4 40.0
2 千岛湖镇 30 8 26.7
3 金峰乡 30 18 60.0
4 临岐镇 30 15 50.0
5 宋村乡 20 8 40.0
6 文昌镇 10 0 0
7 安阳乡 10 4 40.0
8 里商乡 15 0 0
9 鸠坑乡 18 0 0
10 富文乡 15 3 20.0
11 浪川乡 10 0 0
12 姜家镇 20 0 0
13 左口乡 15 0 0
14 瑶山乡 10 3 30.0
15 屏门乡 10 2 20.0
16 枫树岭镇 30 6 20.0
17 界首乡 15 4 26.7
表1  中蜂样品采集信息及蜂房球菌检测结果
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2 张其安,王娟,杨少波 .蜜蜂细菌性疾病及其防治的研究进展.中国蜂业,2011,62:25-30.
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3 卢焕仙,夏培康 .蜜蜂欧洲幼虫腐臭病的发生原因及防治措施.农村实用技术,2009(3):54-55.
LU H X , XIA P K . Occurrence causes and control measures of European foulbrood. Rural Practical Technology, 2009(3):54-55. (in Chinese)
4 BAILEY L , BALL B V . Honey Bee Pathology. 2nd ed. London, UK: Academic Press, 1991.
5 GOVAN V A , ALLSOPP M H , DAVISON S . A PCR detection method for rapid identification of Paenibacillus larvae . Applied Environmental Microbiology, 1999,65(5):2243-2245.
6 周婷,冯峰,董秉义 .中华蜜蜂的欧洲幼虫腐臭病病原研究.昆虫学报,2000,43(增刊1):104-108.
ZHOU T , FENG F , DONG B Y . Study on the pathogen of European larvae rot disease of Chinese honeybee. Acta Entomologica Sinica, 2000,43(Suppl. 1):104-108. (in Chinese)
7 HORNITZKY M A Z , WILSON S C . A system for the diagnosis of the major bacterial brood diseases of honeybees. Journal of Apicultural Research, 1989,28(4):191-195.
8 OMAR R E M , Al E . Isolation and identification of American and European foulbrood from honeybee Apis mellifera (L.). Bulletin of the Entomological Society of Egypt, 2007,85(1):243-256.
9 PINNOCK D E , FEATHERSTONE N E . Detection and quantification of Melissococcus pluton infection in honeybee colonies by means of enzyme-linked immunosorbent assay. Journal of Apicultural Research, 2015,23(3):168-170.
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