Fluorescence-enhanced isothermal amplification for multiplex pathogen detection: Emerging strategies and persistent challenges

Abstract

Multiplex pathogen detection is essential for addressing the increasing complexity of infectious diseases and co-infections, yet conventional polymerase chain reaction-based methods remain hindered by the requirement for sophisticated laboratory infrastructure. Isothermal amplification offers a robust alternative for point-of-care testing, though high-order multiplexing in these systems has historically been limited by signal discrimination challenges. In this review, we synthesize recent breakthroughs in fluorescence-enhanced isothermal amplification, which have transformed multiplex diagnostics through advanced signal encoding and intelligent processing. We illustrate how the convergence of diverse molecular engines, ranging from sequence-specific probes to orthogonal clustered regularly interspaced short palindromic repeats-associated effectors and programmable DNA logic circuits, enables precise, multi-target identification in a single reaction. Beyond the biochemical framework, we further discuss the integration of these molecular strategies with microfluidic platforms, portable optical detection systems, and deep learning-based signal analysis, which collectively facilitate the transition from laboratory prototypes to automated diagnostic platforms. Finally, we examine persistent challenges, including assay crosstalk and amplification bias, and outline future directions toward accessible and scalable multiplex diagnostic systems.