4. Discussion
In the past decades, frequent outbreaks of infectious diseases caused by respiratory viruses have posed a great threat to human health. In order to prevent and control infectious disease epidemics as soon as possible, rapid and accurate diagnostic methods are essential to identify the pathogens. The use of PCR is limited by the need for complex and time-consuming thermal cycling processes, expensive testing equipment and specialized laboratory conditions, so it is not suitable for grassroots and resource-poor areas. In this work, we established a simple, rapid, highly sensitive and specific assay based on fluorescent RT-RAA for the detection of six respiratory viruses (RSVA, Flu A, Flu B, HPIV, SARS-CoV-2 and ADV), each of which could be completed within 20 min at a mild reaction temperature of 39°C. The sensitivity of the RAA assay was tested at 102 copies/mL for RSVA, 102 copies/mL for HPIV, 102copies/mL for Flu A, 103 copies/mL for Flu B, 102 copies/mL for SARS-CoV-2, and 101 copies/mL for ADV, and was confirmed by cross-testing with 21 different pathogen nucleic acids with good specificity. Although the results of parallel testing of pharyngeal swab samples by the RAA method were consistent with those of RT-qPCR, the results obtained by the RAA method within 20 min were more time-saving compared with the 1.5 h required by PCR, because the reaction conditions were mild. Moreover, the required equipment by RT-RAA in this work was more portable (only 2 kg) than that usually required by RT-PCR, and the RT-RAA detector had a built-in battery, which did not require additional power supply and could be carried to the field for testing.
In addition, different from PCR, which requires different heating and cooling processes for the amplification of different pathogenic nucleic acids, RT-assay established in this work can achieve the amplification and detection of six respiratory virus nucleic acids using the same thermostatic procedure. Therefore, in field detection, 16 separate channels of RT-RAA portable detector can be used to flexibly choose single pathogen detection or multiple pathogen detection for test samples. For example, 14 samples (in addition to negative and positive controls) can be tested for single pathogen, or the same sample can be added to different channels for multiple detection of 1 to 6 respiratory viruses at the same time.