title: A closed-tube detection of loop-mediated isothermal amplification (LAMP) products using a wax-sealed fluorescent intercalator.
abstract: LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.
authors.affiliation: College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liang
authors.forename: Sijia
authors.initials: S
authors.lastname: Cheng
authors.forename: Yanan
authors.initials: Y
authors.lastname: Chu
authors.forename: Haiping
authors.initials: H
authors.lastname: Wu
authors.forename: Binjie
authors.initials: B
authors.lastname: Zou
authors.forename: Huan
authors.initials: H
authors.lastname: Huang
authors.forename: Tao
authors.initials: T
authors.lastname: Xi
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D005453:Fluorescence; D057230:Limit of Detection
chemical_list: D017931:DNA Primers; D004247:DNA