title: Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii.
abstract: Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type β-lactamases, are among the leading causes of nosocomial infections worldwide. Among the β-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60°C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of β-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.
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), Harbin Medical University, 157 Baojian Road, Harbin 150081, 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; Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 6348521, Japan. Electronic address: rnakano@naramed-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; Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 6348521, Japan.
authors.forename: Akiyo
authors.initials: A
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: 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: 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: 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: Go
authors.initials: G
authors.lastname: Kamoshida
authors.affiliation: Department of Infection Control and Laboratory Diagnostics, Internal Medicine, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Endo
authors.affiliation: Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 6348521, Japan.
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Yano
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: D000151:Acinetobacter Infections / Q000175:diagnosis / Q000382:microbiology; D040981:Acinetobacter baumannii / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D015780:Carbapenems / Q000494:pharmacology; D003428:Cross Infection / Q000175:diagnosis / Q000382:microbiology; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004352:Drug Resistance, Microbial / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D001618:beta-Lactamases
keywords: Actinetobacter baumanni; Diagnostics; LAMP; Resistance