Figure 4. Sensitivity of the RT-RAA detection system. (A) Fluorescence-time curve for detection of serial 10-fold dilutions of RSVA RNA standards (104-100 copies/ml). (B) HPIV RNA standards (105-100 copies/ml). (C) Flu A RNA standards (105-100 copies /mL). (D) Flu B RNA standards (105-100 copies/mL). (E) SARS-Cov-2 RNA standards (105-100 copies/ml). (F) ADV DNA (105-100 copies/mL). The detection limit of each pathogen can be detected by repeating 3 times.
3.3 Specificity evaluation
To determine the specificity of the assay, 21 different pathogen nucleic acids were investigated in the experiment, including RSVA, RSVB, HPIV, Flu A, Flu B, SARS-CoV-2, ADV, rhinovirus type A, rhinovirus type B, rhinovirus type C, H7N9, HCoV-OC43, HCoV-229E, HCoV-NL63,S. aureus , Chlamydia pneumoniae, Mycoplasma pneumoniae, HMPV, SARS-CoV-2, MERS, and Klebsiella pneumoniae mock sample nucleic acids were used as templates to validate the 6 sets of primers and probes. As shown in Figure 5, except for the detection curves of target pathogen, other curves of non-target pathogens were all negative. It indicated there was no cross-reactivity between different pathogens, reflecting good specificity.