pH-Responsive 1,8-Naphthalimide Probe for a One-Step Loop-Mediated Isothermal Amplification Assay

Loop-mediated isothermal amplification (LAMP) has garnered significant interest as a promising technique for nucleic acid testing, especially in point-of-care and on-site applications, due to its rapid assay speed and simple operating conditions. However, the wide adoption and acceptance of LAMP technology remain a challenge, primarily due to the insufficient sensitivity, poor stability, and strong subjectivity in the interpreting color transition stage. To address these, we developed a novel fluorescent probe, namely, FC-NAPH-PZ, for the LAMP assay. Its high Stokes shift and excellent pH reversibility generated a large and stable fluorescence "turn-on" signal, enabling the highly sensitive (single-copy) and ultrahigh selective detection in the LAMP assay. Meanwhile, its high stability minimized interference with efficient amplification in closed-tube LAMP. Using this probe, we achieved a highly sensitive and specific one-step detection on lambda DNA and Escherichia coli O157:H7 through either visual inspection or quantitative real-time fluorescence monitoring.