COVID-19 was officially declared a global pandemic disease on 11 March 2020, with severe implications for healthcare systems, economic activity, and human life worldwide. Fast and sensitive amplification of the severe acute respiratory syndrome virus 2 (SARS-CoV-2) nucleic acids is critical for controlling the spread of this disease. Here, a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay, targeting conserved positions in the nucleocapsid protein gene (N gene) of SARS-CoV-2, was successfully established for SARS-CoV-2. The assay was specific to SARS-CoV-2, and there was no cross-reaction with other important viruses. The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability. Furthermore, 100% concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 72 clinical specimens. Further linear regression analysis indicated a significant correlation between the real-time RT-RAA and RT-qPCR assays with an R2 value of 0.8149 (p < 0.0001). In addition, the amplicons of the real-time RT-RAA assay could be directly visualized by a portable blue light instrument, making it suitable for the rapid amplification of SARS-CoV-2 in resource-limited settings. Therefore, the real-time RT-RAA method allows the specific, sensitive, simple, rapid, and reliable detection of SARS-CoV-2.

Orf is an important zoonotic disease caused by the Orf virus (ORFV) which can cause contagious pustular dermatitis in goats and sheep. Orf is widespread in most sheep-raising countries in the world, causing huge economic losses. Although diagnostic methods for ORFV infection already exist, it is still necessary to develop a time-saving, labor-saving, specific, low-cost and visual diagnostic method for rapid detection of ORFV in the field and application in grassroots laboratories. This study establishes a DNA extraction–free, real-time, visual recombinase–aided amplification (RAA) method for the rapid detection of ORFV. This method is specific to ORFV and does not cross-react with other common DNA viruses. The detection limits of the real-time RAA and visual judgment of the RAA assay at 95% probability were 13 and 21 copies per reaction for ORFV, respectively. Compared with qPCR, the sensitivity and specificity of the real-time RAA assay were 100%, and those of the visual RAA assay were 92.31% and 100.0%, respectively. The DNA extraction–free visual detection method of RAA established in this study can meet the needs of rapid onsite detection and grassroots laboratories and has important reference value and significance for the early diagnosis of diseased animals.