Development of Duplex PCR Assay for Detection and Differentiation of Typicaland Atypical Melissococcus plutonius strains

Journal of Veterinary Medical Science

Rie ARAI⁴  Masatoshi OKURA⁷  Meihua WU⁸  Yuiko MORINAGA²  Kayo OKUMURA³  Mikio YOSHIYAMA⁵  Tohru MIYOSHI-AKIYAMA¹  Yuya SUGIMURA⁵  Teruo KIRIKAE¹  Daisuke TAKAMATSU⁶ 

[1] Department of Infectious Diseases, National Center for Global Health and Medicine, 1�?21�?1 Toyama, Shinjuku-ku, Tokyo 162�?8655, Japan;Fukuoka-Chuo Prefectural Institute of Animal Health, 4�?14�?5 Hakozakifuto, Higashi-ku, Fukuoka, Fukuoka 812�?0051, Japan;Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, 11 Nishi 2-sen, Inada-cho, Obihiro, Hokkaido 080�?8555, Japan;Saitama Prefectural Chuo Livestock Hygiene Service Center, 107�?1 Besshocho, Kita-ku, Saitama, Saitama 331�?0821, Japan;Honey Bee Research Unit, Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, 2 Ikenodai, Tsukuba, Ibaraki 305�?0901, Japan;The United Graduate School of Veterinary Sciences, Gifu University, 1�?1 Yanagido, Gifu, Gifu 501�?1193, Japan;Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 3�?1�?5 Kannondai, Tsukuba, Ibaraki 305�?0856, Japan;Graduate School of Life and Environmental Sciences, University of Tsukuba, 1�?1�?1 Tennodai, Tsukuba, Ibaraki 305�?8572, Japan

关键词: atypical strain;    duplex PCR;    European foulbrood;    Melissococcus plutonius;    typical strain;   

DOI  :  10.1292/jvms.13-0386

学科分类：兽医学

来源: Japanese Society of Veterinary Science

【 摘 要 】

References(27)Cited-By(3)Melissococcusplutonius is the causative agent of an important honeybee disease, Europeanfoulbrood (EFB). In addition to M. plutonius strains with typicalcharacteristics (typical M. plutonius), we recently reported the presenceof atypical M. plutonius, which are phenotypically and geneticallydistinguished from typical M. plutonius. Because typical and atypicalM. plutonius may have different pathogenic mechanisms, differentiationof these two types is very important for diagnosis and more effective control of EFB. Inthis study, therefore, a duplex PCR assay was developed to detect and differentiatetypical and atypical M. plutonius rapidly and easily. On the basis of theresults of comparative genomic analyses, we selected Na+/H+antiporter gene and Fur family transcriptional regulator gene as targets for detection oftypical and atypical strains, respectively, by PCR. Under optimized conditions, the duplexPCR system using the designed primers successfully detected and differentiated all typicaland atypical M. plutonius strain/isolates tested, while no product wasgenerated from any other bacterial strains/isolates used in this study, including thoseisolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies ofchromosome/reaction for both types, and it could detect typical and atypical M.plutonius directly from diseased honeybee larvae. Moreover, the duplex PCRdiagnosed mixed infections with both M. plutonius types more preciselythan standard culture methods. These results indicate that the duplex PCR assay developedin this study is extremely useful for precise diagnosis and epidemiological study ofEFB.

【 授权许可】

Unknown