title: Molecular detection of Mycoplasma haemomuris subspecies using dnaK-targeted real-time PCR with SYBR Green I and melting curve analysis.
abstract: Hemoplasmas cause severe infections in mammals, but these pathogens are difficult to detect and identify at the species and subspecies level because of the need for time-consuming sequence based methods. Here, we used real-time PCR with SYBR Green I targeting of the dnaK gene followed by standard melting curve analysis to achieve rapid detection and differentiation of the Mycoplasma haemomuris subspecies 'Candidatus Mycoplasma haemomuris subsp. musculi' and 'Candidatus M. haemomuris subsp. ratti'. The melting temperatures of the PCR products, 84.63 ± 0.14 °C for 'Candidatus M. haemomuris subsp. musculi', and 80.72 ± 0.16 °C for 'Candidatus M. haemomuris subsp. ratti', provided clear differentiation between them. Murine hemoplasma DNA samples, which were used as references, were confirmed for species by an analysis of 16S rRNA sequences. The protocol described herein provides a new rapid detection and identification method suitable for use with two recognized subspecies of M. haemomuris.
authors.affiliation: Cooperative Department of Veterinary Medicine, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan. Electronic address: satohsss@iwate-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Satoh
authors.affiliation: Cooperative Department of Veterinary Medicine, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan.
authors.forename: Daiki
authors.initials: D
authors.lastname: Imai
authors.affiliation: Cooperative Department of Veterinary Medicine, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Sato
authors.affiliation: The Iwate Research Center for Wildlife Diseases, 1-17-4 Nakano, Morioka, Iwate, 020-0816, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Furuhama
authors.affiliation: The Iwate Research Center for Wildlife Diseases, 1-17-4 Nakano, Morioka, Iwate, 020-0816, Japan.
authors.forename: Ryô
authors.initials: R
authors.lastname: Harasawa
mesh_terms: D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers / Q000378:metabolism; D003959:Diamines; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D009174:Mycoplasma / Q000235:genetics* / Q000302:isolation & purification*; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals / Q000378:metabolism*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D018840:HSP70 Heat-Shock Proteins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: Hemoplasma; Melting curve; Mycoplasma haemomuris; Rodents; Subspecies