AbstractBackgroundPolymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase‐aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology.MethodsIn this study, we developed and validated the multiplex reverse transcription recombinase‐aided PCR (multiplex RT‐RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process.ResultsUsing recombinant plasmids, the sensitivity of multiplex RT‐RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT‐RAP showed no cross‐reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT‐RAP and the results were found to be consistent with those of corresponding RT‐qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT‐RAP was two to eightfold higher than that of corresponding RT‐qPCR.ConclusionWe conclude the multiplex RT‐RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.