A dual-position nucleic acid coding strategy for one-pot recognition of a pathogen and multiple drug-resistant mutations

Timely and accurate detection of a pathogen and its drug-resistance mutations is crucial for guiding clinical treatment and reducing therapy duration. Traditional methods such as PCR, NGS, and microbial culture, though commonly used, have limitations like slow processing, high costs, and limited sensitivity, making them less suitable for urgent clinical needs. Here we propose a dual-position nucleic acid coding strategy, termed Iso-D-nCir, which can achieve one-pot detection of both conserved region and drug-resistant mutations of a pathogen rapidly and precisely. This approach integrates loop-mediated isothermal amplification (LAMP) and catalytic hairpin assembly (CHA) as the isothermal amplification (Iso) and circuit (Cir) to form the LAMP-D-nCir platform as a model, which is low cost, flexibility of use and short detection time. Using Mycoplasma pneumoniae (MP) and its drug-resistant mutations (A2063G and A2064G) as model targets, LAMP-D-nCir demonstrates ultrahigh sensitivity (200 copies/mL) and specificity. Clinical validation across 126 samples demonstrated 100 % concordance with NGS, PCR, and microbial culture. Additionally, the platform exhibits high-resolution discrimination of low-frequency mutations, 1 % mutation abundance from the mixtures can be detected. Furthermore, CHA enables transduction of analyte into diverse signal outputs, such as electrochemical and colorimetric readouts, thereby rendering Iso-D-nCir particularly well-suited for on-site detection in resource-limited environments.