Carbon nanoparticle-based lateral flow assay for the detection of specific double-tagged DNA amplicons of Paracoccidioides spp.

To overcome the limitations of current methods for diagnosing paracoccidioidomycosis (PCM), it is critical to develop novel diagnostic strategies that can be implemented in low-resource settings and dramatically improve turnaround times. This study focused on the development of a portable molecular test to screen for Paracoccidioides spp. The proposed approach integrated double-tagging polymerase chain reaction (PCR) and a paper-based lateral flow assay (LFA) for readout, using carbon nanoparticles as a signal generation system. Primers tagged with biotin and digoxigenin were employed to conduct the double-tagging PCR, which can be conveniently carried out on portable thermocyclers. This method can generate billions of tagged DNA copies from a single target molecule, which can be rapidly detected by the LFA platform, providing results within minutes. Avidin-modified carbon nanoparticles served as a signal generation system, enabling detection in the immunochromatographic assay. The LFA demonstrated the capability to detect double-tagged amplicons as low as 0.21 ng or 0.10 ng, depending on whether the results were assessed visually or with a smartphone equipped with an image processor. These findings suggest that the proposed approach holds great promise as a point-of-care diagnostic tool for the early and accurate detection of PCM in low-resource settings. The diagnostic test is rapid and inexpensive, requires minimal handling and can be easily introduced into the general practitioner's armoury for ambulatory screening of infection. This innovative approach has the potential to make a substantial contribution to PCM diagnosis, ultimately reducing morbidity and mortality associated with this disease.