title: Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains.
abstract: Melissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na(+)/H(+) antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.
authors: Saitama Prefectural Chuo Livestock Hygiene Service Center, 107-1 Besshocho, Kita-ku, Saitama, Saitama 331-0821, Japan.
authors: Rie
authors: R
authors: Arai
authors: Tohru
authors: T
authors: Miyoshi-Akiyama
authors: Kayo
authors: K
authors: Okumura
authors: Yuiko
authors: Y
authors: Morinaga
authors: Meihua
authors: M
authors: Wu
authors: Yuya
authors: Y
authors: Sugimura
authors: Mikio
authors: M
authors: Yoshiyama
authors: Masatoshi
authors: M
authors: Okura
authors: Teruo
authors: T
authors: Kirikae
authors: Daisuke
authors: D
authors: Takamatsu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001516:Bees / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D056569:Enterococcaceae / Q000235:genetics*; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis*; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics; D013045:Species Specificity; D015534:Trans-Activators / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D017923:Sodium-Hydrogen Exchangers; D015534:Trans-Activators