Open AccessDetection of periodontal pathogen Porphyromonas gingivalis by loop‐mediated isothermal amplification method
Author(s) -
Maeda Hiroshi,
Kokeguchi Susumu,
Fujimoto Chiyo,
Tanimoto Ichiro,
Yoshizumi Wakako,
Nishimura Fusanori,
Takashiba Shogo
Publication year - 2005
Publication title -
fems immunology & medical microbiology
Language(s) - English
Resource type - Journals
eISSN - 1574-695X
pISSN - 0928-8244
DOI - 10.1016/j.femsim.2004.08.005
Subject(s) - loop mediated isothermal amplification , porphyromonas gingivalis , sybr green i , naked eye , amplicon , biology , agarose gel electrophoresis , microbiology and biotechnology , detection limit , periodontal pathogen , nucleic acid , 16s ribosomal rna , polymerase chain reaction , bacteria , dna , chemistry , chromatography , gene , biochemistry , genetics
A method for nucleic acid amplification, loop‐mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis . A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 °C with a detection limit of 20 cells of P. gingivalis . Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked‐eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by‐product of LAMP) in the tube. Detection limits of these naked‐eye inspections were 20 cells and 200 cells, respectively. Although false‐positive DNA amplification was observed from more than 10 7 cells of Porphyromonas endodontalis , no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real‐time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10 2 –10 6 cells. The real‐time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real‐time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis , especially in its rapidity and simplicity.