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 10² 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.