A rapid and label-free DNA-based interference reduction nucleic acid amplification strategy for viral RNA detection

Common reference methods for COVID-19 diagnosis include thermal cycling amplification (e.g. RT-PCR) and isothermal amplification methods (e.g. LAMP and RPA). However, they may not be suitable for direct detection in environmental and biological samples due to background signal interference. Here, we report a rapid and label-free interference reduction nucleic acid amplification strategy (IR-NAAS) that exploits the advantages of luminescent iridium(III) probes, time-resolved emission spectroscopy (TRES) and multi-branch rolling circle amplification (mbRCA). Using IR-NAAS, we established a luminescence approach for diagnosing COVID-19 RNAs sequences RdRp, ORF1ab and N with a linear range of 0.06-6.0 × 10⁵ copies/mL and a detection limit of down to 7.3 × 10⁴ copies/mL. Moreover, the developed method was successfully applied to detect COVID-19 RNA sequences from various environmental and biological samples, such as domestic sewage, and mice urine, blood, feces, lung tissue, throat and nasal secretions. Apart from COVID-19 diagnosis, IR-NAAS was also demonstrated for detecting other RNA viruses, such as H1N1 and CVA10, indicating that this approach has great potential approach for routine preliminary viral detection.