Development of a duplex real-time recombinase aided amplification assay for the simultaneous and rapid detection of PCV3 and PCV4

Figures

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  Fig. 1. Positions of the duplex real-time RAA primers and probes in the ORF2 (Cap gene) sequences of different PCV3 strains in the GenBank database. Dots represent nucleotide residues that match the majority. The forward primers are marked with red bars, the reverse primers are marked with blue bars, and probes are marked with green bars

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  Fig. 2. Positions of the duplex real-time RAA primers and probes in the ORF1 (Rep gene) sequences of different PCV4 strains in the GenBank database. Dots represent nucleotide residues that match the majority. The forward primers are marked with red bars, the reverse primers are marked with blue bars, and probes are marked with green bars

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  Fig. 3. Positions of the duplex real-time RAA primers and probes in the ORF2 (Cap gene) sequences of different PCV4 strains in the GenBank database. Dots represent nucleotide residues that match the majority. The forward primers are marked with red bars, the reverse primers are marked with blue bars, and probes are marked with green bars

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  Fig. 4. Screening the optimal primer-probe combination for the duplex real-time RAA for PCV3 and PCV4. Representative amplification results of the duplex real-time RAA assay using 6 primer-probe combinations. Amplification curves in different colors represent different primer-probe combinations. (A) The amplification result of the FAM channel, corresponding to the amplification curves of PCV4; (B) The amplification result for the HEX channel, corresponding to the amplification curves of PCV3; (C) The amplification results of both channels

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  Fig. 5. Optimization of primer pairs and probes concentrations for the duplex real-time RAA assay of PCV3 and PCV4. Amplification curves in different colors represent different concentration combinations of primer pairs or different concentrations of probes. The amplification result of the FAM channel, corresponding to the amplification curves of PCV4. The amplification result for the HEX channel, corresponding to the amplification curves of PCV3. (A-C) Representative amplification results of the duplex real-time RAA assay using 6 concentration combinations of primer pairs; (D-F) Representative amplification results of the duplex real-time RAA assay using 5 concentrations of probes

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  Fig. 6. Specificity and sensitivity analysis of the duplex real-time RAA assay for PCV3 or PCV4. (A) Analytical specificity test of the duplex real-time RAA assay. Amplification curves in blue corresponding to the FAM channel, and those in green corresponding to the HEX channel. Curves 1: pUC57-PCV3 and pUC57-PCV4, 2: pUC57-PCV3, 3: pUC57-PCV4, 4–13: nucleic acids of FMDV, CSFV, PRV, PPV, PCV2, PEDV, TGEV, PRRSV, PDCoV, and the negative control, respectively; (B) Analytical sensitivity test of the duplex real-time RAA assay. Amplification curves in blue corresponding to the FAM channel, and those in green corresponding to the HEX channel. Curves 1–9, correspond to 107-100 copies and the negative control, respectively; (C) Probit regression analysis of the duplex real-time RAA assay results, the detection limit of the duplex real-time RAA assay at 95% reliability (73.67 copies/reaction) is marked with a red number