Visual detection of Coxsackievirus A6 using a reverse transcription polymerase spiral reaction method

Figures

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  Figure 1. Analysis of the products after amplification with the indicated primers. Electrophoresis was performed on a 1.5% agarose gel to analyze the amplification products of RT-PSR assays with six different sets of primers. Lanes 1-6 indicate primers no. 1 to 6 respectively. Lane NC, negative control (RNase-Free ddH2O); Lane M, DL 2000 DNA marker.

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  Figure 2. Key reaction parameters for the RT-PSR assay, including primer concentration, reaction temperature, and duration, were optimized using CVA6 RNA. (A) Analysis of the products after amplification with primer 1 at the indicated primer concentrations. Electrophoresis was performed on a 1.5% agarose gel to analyze the amplification products of RT-PSR assays with primer 1 at the primer concentrations of 1.6 µmol/L (Lane 1); 2.0 µmol/L (Lane 2), 2.4 µmol/L (Lane 3), 2.8 µmol/L (Lane 4), 3.2 µmol/L (Lane 5), and 3.6 µmol/L (Lane 6), respectively. Lane NC, negative control (RNase-Free ddH2O); Lane M, DL 2000 DNA marker. (B) Analysis of the product after amplification with primer 1 at the indicated temperatures. RT-PSR amplification was carried out at the temperatures of 59°C (Lane 1), 59.6°C (Lane 2), 60.8°C (Lane 3), 62.4°C (Lane 4), 64.5°C (Lane 5), 66.2°C (Lane 6), 67.3°C (Lane 7), and 68.0°C (Lane 8), respectively. Lane M, DL 2000 DNA marker. (C) Analysis of the product after amplification with primer 1 at 64.5°C for the indicated time periods. RT-PSR amplification was performed for 30 min (Lane 1), 40 min (Lane 2), 50 min (Lane 3), and 60 min (Lane 4), respectively. Lane M, DL 2000…

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  Figure 3. Sensitivity of the RT-PSR amplification for CVA6. (A) Visual detection of RT-PSR amplification products utilizing the HNB indicator. Tube 1, 1 × 108 copies/µL; Tube 2, 1 × 107 copies/µL; Tube 3, 1 × 106 copies/µL; Tube 4, 1 × 105 copies/µL; Tube 5, 1 × 104 copies/µL; Tube 6, 1 × 103 copies/µL; Tube 7, 1 × 102 copies/µL; Tube 8, 1 × 101 copies/µL; NC, negative control (DEPC-treated water). (B) Agarose gel electrophoresis of RT-PSR amplification products. Lane M: DL 1,000 DNA marker. Lane 1, 1 × 108 copies/µL; Lane 2, 1 × 107 copies/µL; Lane 3, 1 × 106 copies/µL; Lane 4, 1 × 105 copies/µL; Lane 5, 1 × 104 copies/µL; Lane 6, 1 × 103 copies/µL; Lane 7, 1 × 102 copies/µL; Lane 8, 1 × 101 copies/µL; NC, negative control (RNase-Free ddH2O). (C) Visual detection of RT-PSR amplification products. Tube 1, hepatitis B virus; Tube 2, hepatitis C virus; Tube 3, respiratory syncytial virus; Tube 4, cytomegalovirus; Tube 5, EV71; Tube 6, CVA10; Tube 7, CVA16; Tube 8, CVA6; Tube 9, rotavirus; NC, negative control (RNase-Free ddH2O). (D) Agarose gel electrophoretic profile of RT-PSR amplification products from 9 viral strains. Lane M: DL 2,000 DNA marker. Lane 1, hepatitis B virus; Lane 2,…

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  Figure 4. The results of visualized RT-PSR to detect clinical samples. Visual detection of RT-PSR amplification products from clinical samples (tube no. 1 to 142). NC, negative control (RNase-FreeH2O); Pos, 108 copies/µL the standard plasmid.