Colorimetric and fluorescent dual-modality assay for cell-free mitochondrial DNA copy number in saliva

Copy number of cell-free mitochondrial DNA (cf-mtDNA) has garnered significant attention as a biomarker for studying and diagnosing various diseases. However, Quantitative Real-time PCR (qPCR) and Droplet Digital PCR (ddPCR) assays for cf-mtDNA copy number detection require expensive equipment and high experiment conditions. In this study, a colorimetric and fluorescent dual-modality assay was developed for quantitative detection of cf-mtDNA copy number. With G-quadruplex (G4) sequence modified primers, the assay could quantitatively detect cf-mtDNA with spectrophotometry, RGB (Red, Green, Blue) visual method and fluorescence method, which made the application scenarios more diverse. The specificity of dual-mode method was better, and the detection limits of spectrophotometry, RGB visual method and fluorescence method were as low as 1.45 × 10^-1 copies/μL, 1.65 copies/μL and 1.58 × 10^-1 copies/μL, respectively. Compared with qPCR and ddPCR assays developed in previous studies, the dual-modality assay in this study had a lower detection limit. It was also independent of expensive qPCR and ddPCR equipment and the detection cost was low. Therefore, the colorimetric and fluorescent dual-modality assay represent a label-free and sensitive approach for assessing cf-mtDNA levels, offering promising implications for biomedical research and clinical diagnostics.