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 x 10(5) copies/mL and a detection limit of down to 7.3 x 10(4 )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.

Automated summary

In this study, a rapid and label-free interference reduction nucleic acid amplification strategy (IR-NAAS) was developed for diagnosing COVID-19 RNAs sequences. It utilized luminescent iridium(III) probes, time-resolved emission spectroscopy (TRES), and multi-branch rolling circle amplification (mbRCA) to achieve high sensitivity and specificity. The method was successfully applied to detect COVID-19 RNA sequences in various environmental and biological samples, and also showed potential for detecting other RNA viruses.