Fig. 3.

The amplification curves of the duplex real time RT-RAA assays using 10-fold dilution series of plasmid DNA containing the VP1gene of EV 71 (a) and CA16 (b). Fluorescence signals from target amplification were recorded in the FAM detection channels of the QT-RAA1620 device, while IAC amplification was recorded in the HEX detection channel. The development of fluorescence using a dilution range of 10⁶ copies/μL − 1 copy/μL of the target recombinant plasmid standards. a represents the duplex RT- RAA of EV71 with 50copies IAC plasmid per reaction. The sensitivity of the assay is 10 copies. High concentrations of target plasmids (10⁶copies/μL) completely inhibit the amplification of IAC plasmid. EV71 amplification curves of high concentration plasmid (10⁶ copies per reaction) occurred early and even overlapped, while for the IAC, no amplification curves were observed in the HEX channel. EV71 amplification curves of medium and low concentration plasmids appeared slightly later than the high concentration, while for the IAC, amplification curves were observed in the HEX channel. b represents the duplex RT-RAA of CA16 with 100 copies IAC plasmid per reaction. The sensitivity of the assay is 10 copies. High concentrations of target plasmids (10⁶-10⁵copies/μL) completely inhibit the amplification of IAC plasmid. The amplification curves of CA16 plasmids with high concentration (10⁶ -10⁵copies per reaction) appeared early and even overlapped, while for the IAC, no amplification curves were observed in the HEX channel. CA16 plasmids with medium and low concentrations revealed no significant interference with IAC amplification curves in the HEX channel