Rapid, sensitive, and visual detection of Talaromyces marneffei using multiple cross-displacement amplification coupled with a nanoparticle-based biosensor platform

Talaromyces marneffei (T. marneffei) is an opportunistic pathogenic fungus that causes life-threatening disseminated talaromycosis. Misdiagnosis or delayed diagnosis markedly increases mortality rates, thereby constituting a critical public health concern. Consequently, there is an urgent need for a rapid, sensitive, and highly specific diagnostic strategy. Herein, we developed a novel diagnostic assay termed T. marneffei-multiple cross displacement amplification (MCDA)-lateral flow biosensor (LFB), which integrates the innovative nucleic acid amplification technique known as MCDA with a nanoparticle-based LFB. The assay was developed using ten specific primers targeting the internal transcribed spacer (ITS2) region of the T. marneffei rRNA gene. The optimal amplification conditions were established as 67 °C for 35 min, eliminating the need for complex thermal cycling equipment and requiring only a water bath for amplification. The assay revealed high sensitivity with a limit of detection of 1 fg genomic DNA per reaction. No cross-reaction was detected with non-T. marneffei species, indicating high specificity. The clinical application of this assay was primarily assessed by comparing 100 clinical samples and 35 simulated clinical samples against the culture method. The total detection procedure, encompassing DNA extraction and reagent preparation (25 min), T. marneffei–MCDA reaction (35 min at 67 °C), and LFB detection (2 min), can be completed in 62 min, enhancing detection efficiency. This strategy markedly diminishes dependence on specialized laboratory equipment or professional expertise, rendering it suitable for rapid on-site testing and application within primary healthcare environments.