title: Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection.
abstract: Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at intensive care unit (ICU). A rapid and sensitive detection of VRE infection is in high demand for timely and suitable antibiotic treatment. Here, we optimized a distinct DNA-based diagnostic technique, loop-mediated isothermal amplification (LAMP) for a rapid detection of the presence of vanA gene, a critical component of the gene cluster required for vancomycin resistance. Amplification efficiency was optimal at 62°C and with 2mM MgSO4. The detection limit of the DNA template was 80pg and LAMP amplicons were detected within 40min; thereby suggesting a potential applicability of LAMP as a sensitive and urgent diagnostic method. Furthermore, positive LAMP reaction was directly detected with the naked-eye by monitoring the formation of a white precipitate or the color change induced by hydroxy naphthol blue (HNB) dye. Finally, 56 clinical isolates were successfully tested for the presence of vanA gene by LAMP, which was determined to be more sensitive than PCR. Together, our results clearly demonstrate the usefulness of LAMP for the diagnosis of VRE infection.
authors.affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Yu Jin
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Dong Eun
authors.initials: DE
authors.lastname: Yong
authors.affiliation: Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Kyungwon
authors.initials: K
authors.lastname: Lee
authors.affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jeon Han
authors.initials: JH
authors.lastname: Park
authors.affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul, Republic of Korea. Electronic address: jaemyun@yuhs.ac.
authors.forename: Jae Myun
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul, Republic of Korea. Electronic address: sangsun_yoon@yuhs.ac.
authors.forename: Sang Sun
authors.initials: SS
authors.lastname: Yoon
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D019729:Carbon-Oxygen Ligases / Q000235:genetics*; D003428:Cross Infection / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D065507:Vancomycin-Resistant Enterococci / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; C071437:VanA ligase, Bacteria; D019729:Carbon-Oxygen Ligases
keywords: Diagnosis; Loop-mediated isothermal amplification (LAMP); Vancomycin-resistant enterococci (VRE)