title: Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).
abstract: Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. Electronic address: nakano@med.teikyo-u.ac.jp.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Nakano
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Akiyo
authors.initials: A
authors.lastname: Nakano
authors.affiliation: Department of Microbiology and Infectious Diseases, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Tsuneyuki
authors.initials: T
authors.lastname: Ubagai
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Takane
authors.initials: T
authors.lastname: Kikuchi-Ueda
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Kikuchi
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Tansho-Nagakawa
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Go
authors.initials: G
authors.lastname: Kamoshida
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan; Genomics Research Center (One of the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), 157 Baojian Road, Harbin, Heilongjiang Province, 150081, PR China.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Mu
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ono
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001769:Blood / Q000382:microbiology; D017931:DNA Primers / Q000737:chemistry; D005243:Feces / Q000382:microbiology; D006801:Humans; D007710:Klebsiella Infections / Q000175:diagnosis*; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D013183:Sputum / Q000382:microbiology; D014556:Urine / Q000382:microbiology; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D001618:beta-Lactamases; C531581:carbapenemase-2, Klebsiella pneumoniae
keywords: Antibiotic resistance gene; Klebsiella pneumoniae carbapenemase (KPC); Loop-mediated isothermal amplification; Rapid detection