A Rebuilding‐Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N‐in‐1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens

Abstract

Sensitive, rapid and low‐cost nucleic acid detection is critical for controlling infectious pathogens. Here, we develop a ready‐to‐use and multimodal detection based on a rebuilding‐free, ultrasensitive and selective strategy named dual hairpin ligation‐induced isothermal amplification pro (DHLApro). Taking influenza A, influenza B, MERS‐CoV, SARS‐CoV‐2 as model targets, we demonstrate DHLApro provides ≈zM level ultra‐sensitivity, being equaling to 0.45 copy/μL in original sample. By simply changing the recognition module, a set of DHLApro components can be applied to a new target without performance loss. Moreover, DHLApro innovatively allows flexible logic/multiplex assay using one set of primer, for example, the “N pathogens‐in‐1” OR gate screening and accurate multi‐channel multiplex assay. Compared with traditional methods, the cost of this logic/multiplex assay has been largely reduced and the cross‐interference between the multiple primer sets is also avoided.