Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

Figures

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  Fig. 1. Detection of the RAA amplicons by lateral flow strip. The sample pad contains gold-labeled anti-FAM antibodies, the test line was coated with biotin-ligands, and control line was coated with anti-rabbit antibodies. The double-labeled amplicons (FAM and biotin) were diffused through the chromatographic membrane, and when they diffused to the test line, the products were captured by the biotin -ligands, resulting in an appearance of red-pink color. Non-captured particles will be fixed at the control line by anti-rabbit antibodies. In the absence of target amplicons, color will appear at control line only [26]

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  Fig. 2. Schematic diagram of duplex real time RAA assays for detection of EV71 and CA16

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  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…

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  Fig. 4. Performance of EV71 LFS RT-RAA assay. a Optimization experiment of LFS RT-RAA reaction time. When the reaction time was longer than 20 min, the test line was visible. b Analytical sensitivity of the LFS RT- RAA assay. Lane1, 1 × 106 copies, Lane2, 1 × 105copies, Lane3, 1 × 104copies, Lane4, 1 × 103copies, Lane5, 1 × 102 copies, Lane6, 1 × 101 copies, Lane7, 1 copy, Lane8, no template control. c Analytical specificity of the LFS RT-RAA assay. Only EV71 specimen was amplified. The other samples were not amplified. Lane1, EV71, Lane2, CA 16, Lane3, CA6, Lane4, CA10, Lane5, CA5, Lane6, CA9, Lane7, CA24, Lane8, CB2, Lane9, CB4, Lane10, PV2, Lane11, PV3, Lane12, Eco30, Lane13, HEV14, Lane14, no template control

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  Fig. 5. Performance of CA16 LFS RT-RAA assay. a Optimization experiment of LFS RT-RAA reaction time. When the reaction time was longer than 20 min, the test line was visible. b Analytical sensitivity of the LFS RT-RAA assay. Lane1, 1 × 106 copies, Lane2,1 × 105copies, Lane3, 1 × 104copies, Lane4, 1 × 103copies, Lane5, 1 × 102 copies, Lane6, 1 × 101 copies, Lane7, 1copy, Lane8, no template control. c Analytical specificity of the LFS RT-RAA assay. Only CA16 specimen was amplified. The other samples were not amplified. Lane1, CA16, Lane2, EV71, Lane3, CA6, Lane4, CA10, Lane5, CA5, Lane6, CA9, Lane7, CA24, Lane8, CB2, Lane9, CB4, Lane10, PV2, Lane11,PV3, Lane12, Eco30, Lane13, HEV14, Lane14, no template control