RH-RPA: A Rapid and Highly Sensitive Assay for Nucleic Acid Detection Based on RNase HII Combined with Recombinase Polymerase Amplification

Currently, RPA-exo and RPA-nfo are the primary methods for RPA/RT-RPA probe assays, both of which have been widely applied to the detection of various targets. However, RPA-nfo exhibits lower sensitivity compared with the exo probe method, while RPA-exo lacks the capability for equipment-free visualization inherent to RPA-nfo. Both of the approaches mentioned above limit the broader application of RPA/RT-RPA probe assays. To address those limitations, we have developed a novel recombinase polymerase amplification (RPA) combined with an <i><i>E. coli</i></i> RNase HII assay (RH-RPA). This approach supports both fluorescence signal detection and lateral-flow strip readouts. Due to the high efficiency and specificity of <i><i>E. coli</i></i> RNase HII in recognizing and cleaving targets, this method serves as a rapid and accurate molecular diagnostic platform. Under the fluorescence detection mode, RH-RPA achieves a limit of detection as low as 10 copies per reaction for both DNA and RNA within 20 min. Additionally, the lateral-flow strip mode enables the detection of as few as 5 copies per reaction of nucleic acids within 20 min. In clinical sample analysis, the RT RH-RPA demonstrated 100% accuracy in detecting the influenza A virus, underscoring its reliability in practical diagnostics. These findings highlight the stable specificity, rapid performance, high sensitivity, and cost-effectiveness of the RH-RPA methods, showcasing their potential as promising tools for point-of-care nucleic acid detection.