2.6 Performance verification
2.6.1 Sensitivity evaluation
The sensitivity of the assay was characterized by serial tenfold dilutions of nucleic acid standards (from 105 to 1 RNA/DNA copies/mL), with ddH2O as a negative control and the same system configuration is shown in Table S3.
2.6.2 Specificity evaluation
The specificity of the detection systems of the six pathogen primer probes was evaluated using RSVA, RSVB, HPIV, H1N1, influenza B virus, SARS-CoV-2, adenovirus, rhinovirus type A, rhinovirus type B, rhinovirus type C, H7N9, OC43, 229E, NL63, and S. aureus, Chlamydia pneumoniae, Mycoplasma pneumoniae, HMPV, SARS-CoV-2, MERS, and Klebsiella pneumoniae mock sample nucleic acids, respectively, at a template concentration of 105copies/mL.
2.6.3 Comparison of the consistency of pharyngeal swab results using real-time polymerase chain reaction and RT-RAA
71 positive pharyngeal swab samples were tested by the RT-RAA and PCR method, including 11 syncytial viruses, 7 influenza A viruses, 30 influenza B viruses, 4 parainfluenza virus, 11 novel coronaviruses, and 8 adenoviruses.
Real-time PCR was performed on a Bio-rad instrument, in which a commercial virus detection kit (Shenzhen Aodong Inspection and Testing Technology Co.) was used. Reactions were conducted in a 25 μL volume following kit instructions. RNA was extracted from 71 positive pharyngeal swab samples for method comparison experiments. All samples were tested by two methods, one-tube RT-RAA and real-time PCR assays. The concordance between these two methods was compared using SPSS 24.0 software.