Specific lateral flow detection of isothermal nucleic acid amplicons for accurate point-of-care testing

For point-of-care testing (POCT), coupling isothermal nucleic acid amplification schemes (e.g., recombinase polymerase amplification, RPA) with lateral flow assay (LFA) readout is an ideal platform, since such integration offers both high sensitivity and deployability. However, isothermal schemes typically suffers from non-specific amplification, which is difficult to be differentiated by LFA and thus results in false-positives. Here, we proposed an accurate POCT platform by specific recognition of target amplicons with peptide nucleic acid (PNA, assisted by T7 Exonuclease), which could be directly plugged into the existing RPA kits and commercial LFA test strips. With SARS-CoV-2 as the model, the proposed method (RPA-TeaPNA-LFA) efficiently eliminated the false-positives, exhibiting a lowest detection concentration of 6.7 copies/μL of RNA and 90 copies/μL of virus. Using dual-gene (orf1ab and N genes of SARS-CoV-2) as the targets, RPA-TeaPNA-LFA offered a high specificity (100%) and sensitivity (RT-PCR Ct < 31, 100%; Ct < 40, 71.4%), and is valuable for on-site screening or self-testing during isolation. In addition, the dual test lines in the test strips were successfully explored for simultaneous detection of SARS-CoV-2 and H1N1, showing great potential in response to future pathogen-based pandemics.