Fig. 1

Schematic outline of the iAFA multiplex assay. a Schematic of the iAFA multiplex assay protocol, along with the approximate time required for each step. The assay integrated one-step multiplex RT-PCR and capillary electrophoresis to produce a time-saving and labor-saving method. b The UDG enzyme-based elimination of carryover contaminants by specifically cutting the 5′ side of the dUTP-incorporated amplicon DNA while having no effect on RNA templates. During the multiplex RT-PCR reaction, the possible contaminants were degraded into small fragments, and the UDG enzyme was inactivated at approximately 50 °C, ensuring that only the RNA template was amplified