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.