Antiphasic Arctangent-Arccotangent (AAA) probes: A kinetically orthogonal design for single-tube, real-time, and low-crosstalk multiplex LAMP.

The rapid and discriminative detection of multiple pathogens is crucial for effective disease control, yet remains a significant challenge for point-of-care testing (POCT). Herein, we report a novel probe design strategy-Antiphasic Arctangent-Arccotangent (AAA) probes-for kinetically orthogonal, low-crosstalk multiplex detection in a single closed tube via loop-mediated isothermal amplification (LAMP). The AAA probe system comprises two kinetically complementary probes: the Arctangent Probe (ATP), a stem-loop probe that yields a rising fluorescence signal, and the Arccotangent Probe (ACP), a guanine-quenched linear probe that produces a falling fluorescence signal. Their inherently antiphasic kinetic trajectories effectively reduce false positives arising from nonspecific amplification. This system requires no additional enzymes or complex operations, preserving the simplicity of conventional LAMP. As a proof of concept, we demonstrated its utility by simultaneously detecting Japanese encephalitis virus (JEV) and Plasmodium vivax (P. vivax), two representative mosquito-borne pathogens, with high specificity and a sensitivity of 5 copies/μL. The assay performed robustly with clinical samples and in complex matrices (e.g., serum, tissue homogenates). Beyond the specific duplex detection, the AAA probe principle establishes a general framework for designing orthogonal probe sets, offering a promising and versatile strategy for multiplex nucleic acid detection in resource-limited settings.