Fig. 1

A Schematic diagram of the triplex NALFIA. 5’ labeled duplex target amplicons are generated by RT‒PCR using forward and reverse primers with 5’ tags. The labeled amplicons are then captured through the tags on the forward primer by the corresponding anti-tag/label antibody, which is the test line on the LFA device, while the biotin labeled on the reverse primer helps capture the detector system, i.e., the stretavidin-colloidal gold conjugate, and produces the signal on the test line in the presence of specific amplification. Label 1 - Alexa Fluor 488, Label 2 - Rhodamine Red, Label 3 - Digoxigenin. B Schematic diagram of the NALFIA triplex layout with coated lines, signal development and result interpretation. Signals at test line 1 and the control line indicate a positive test for REBOV and a negative test for NiV and MERS-CoV. Signals at test line 2 and the control line indicate a positive test for MERS-CoV and a negative test for REBOV and NiV. Signals at test line 3 and the control line indicate a positive test for NiV and negative tests for MERS-CoV and REBOV. No signal at the conjugate control line, regardless of signal development on any of the test lines, shows that the test is invalid