Programmable Multisite Recognition Enables Amplification-Free Nanopore Detection of Mycobacterium tuberculosis in Sputum and Plasma.

Tuberculosis diagnostics remain challenged by low target abundance, strain diversity, and the need for reliable testing in difficult clinical specimens. Here, we develop an amplification-free nanopore sensing platform based on programmable linear DNA nanohook arrays for direct detection of Mycobacterium tuberculosis (Mtb) DNA. This work presents a dual-target, multisite recognition strategy that simultaneously captures two conserved insertion sequences, IS6110 and IS1081, and converts their binding events into resolvable nanopore electrical signatures. This design improves target recovery and reduces dependence on any single genomic locus, enabling robust detection across diverse Mtb strains. Using this approach, we achieved a limit of detection of 9.5 copies/μL and demonstrated accurate identification of Mtb in both sputum and plasma samples without nucleic acid amplification. Our results show that programmable multisite recognition combined with nanopore readout provides a powerful route toward sensitive, sample-flexible tuberculosis diagnostics.