Figure 1. Principle diagram of amplification of primers and recombinant enzyme reaction by RAA technique.
Zhang et al. established a method for the fluorescence detection of SARS-CoV-2 clinical samples by RT-RAA, and the results showed that the clinical detection performance of RT-RAA kits was comparable to that of RT-PCR kits, and RT-RAA has the advantages of simple operation and portability, and is considered an important alternative to RT-PCR to meet the needs of community-level medical institutions27. Nie et al. constructed a method for detecting JEV using RT-RAA. It can detect multiple genotypes of JEV, which is simpler and more convenient than the traditional PCR assay. The detection limit of JEV plasmid is 5.5 copies/μL, which is similar to JEV RT-LAMP and TaqMan RT-qPCR, slightly higher than SYBR Green I RT-qPCR, and 100 times higher than RT-PCR. The established JEV RT-RAA fluorescence assay has been used in clinical diagnosis with rapidity, high sensitivity and specificity28.
In this work, we established a rapid, highly sensitive and specific fluorescent RAA assay for six respiratory viruses. The flow chart of the fluorescent RT-RAA experiment is shown in Figure 2. With a portable built-in power supply device, nucleic acid amplification of various pathogens could be completed in one test within 20 minutes at 39℃, and the test results can be observed in real time. This study could provide technical support for the portable, reliable and sensitive detection of multi-pathogen on the infectious disease site.