イマイ　カズオ   IMAI Kazuo   今井　一男    所属   埼玉医科大学  医学部 臨床検査医学（中央検査部）    職種   専任講師
論文種別	学術雑誌(原著)
言語種別	英語
査読の有無	査読あり
表題	A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay
掲載誌名	正式名：JOURNAL OF MICROBIOLOGICAL METHODS ISSNコード：01677012
出版社	ELSEVIER SCIENCE BV
巻・号・頁	141,90-96頁
著者・共著者	Jun Sakai,Takuya Maeda,Norihito Tarumoto,Kazuhisa Misawa,Shinsuke Tamura,Kazuo Imai,Toshiyuki Yamaguchi,Satoshi Iwata,Takashi Murakami,Shigefumi Maesaki
発行年月	2017/10
概要	Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae. This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae.Reactions were performed at 62 degrees C for 60 min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0 fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0 pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures.This method would be a simple
DOI	10.1016/j.mimet.2017.08.009
PMID	28811193