Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria.
Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal cycler, and then by measuring the turbidity of the reaction solution using a real-time turbidimeter. As a result, it was possible to quantify the initial amoA DNA concentration from an environment with a sensitivity down to 10(2) DNA copies of target DNA and a dynamic range of 7-9 orders in magnitude. Background DNA from nontargeted bacteria (Pseudomonas denitrificans) that does not encode amoA gene did not affect the quantitative capability of LAMP. Over results suggested that the real-time LAMP is effective for monitoring microorganisms and their gene expression in environments.
Aoi
Yoshiteru
Y
Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. yoshiteruaoi@toki.waseda.jp
Hosogai
Mariko
M
Tsuneda
Satoshi
S
D000641:Ammonia / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry; D004784:Environmental Monitoring / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics / Q000378:metabolism; D007364:Intercalating Agents / Q000494:pharmacology; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry / Q000379:methods; D043370:Nitrosomonas europaea / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012107:Research Design; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D000641:Ammonia