FIG 1.

Target amplification with the CARDS toxin LAMP assay. (A) Representative amplification of serial dilutions of M. pneumoniae nucleic acid using the LAMP assay targeting the CARDS toxin gene. LAMP reactions with calcein were monitored in real-time on the FAM channel of an ABI 7500 instrument. As the less-abundant Mn²⁺ precipitates from the reaction with a pyrophosphate ion, Mg²⁺ binds to calcein, and it fluoresces upon excitation with 495-nm-wavelength light. A no-template control reaction (NTC) served as the baseline. ΔRn is defined as the magnitude of the fluorescence signal generated during the PCR at each time point. (B) Restriction digest with MboI, which recognizes a site within the M. pneumoniae CARDS toxin amplicon, cut all products into the expected identical component parts, thereby confirming specificity. 1, Bioanalyzer ladder; 2, LAMP amplicon species, 3, LAMP amplicons digested with MboI.