RT-nestRPA is a new technology for the rapid and sensitive detection of nucleic acid detection of pathogens used for a variety of medical application scenarios.

BACKGROUND: The effective detection of pathogens is of great importance for the diagnosis and treatment of infectious diseases. We have proposed the novel RT-nestRPA technique for SARS-CoV-2 detection, which is a rapid RNA detection technique with ultra-high sensitivity. RESULTS: The RT-nestRPA technology has a sensitivity of 0.5 copies/uL of synthetic RNA targeting the ORF7a/7b/8 gene or 1 copy/uL synthetic RNA targeting the N gene of SARS-CoV-2. The entire detection process of RT-nestRPA only takes only 20 min, which is significantly shorter than RT-qPCR (nearly 100 min). Additionally, RT-nestRPA is capable of detecting dual genes of SARS-CoV-2 and human RPP30 simultaneously in one reaction tube. The excellent specificity of RT-nestRPA was verified by analyzing twenty-two SARS-CoV-2 unrelated pathogens. Furthermore, RT-nestRPA had great performance in detecting samples treated with cell lysis buffer without RNA extraction. The innovative double-layer reaction tube for RT-nestRPA can prevent aerosol contamination and simplify the reaction operation. Moreover, the ROC analysis revealed that RT-nestRPA had high diagnostic value (AUC = 0.98), while the AUC of RT-qPCR was 0.75. SIGNIFICANCE: Our current findings suggested that RT-nestRPA could serve as a novel technology for nucleic acid detection of pathogens with rapid and ultrahigh sensitive features used in various medical application scenarios.