Figure 3

Sensitivity, specificity analysis of the real-time RT-PCR and clinical samples detection. A: Real-time RT-PCR amplification plot of the CVA6 viral gene segment. A PCR baseline subtractive curve fit view of the data is shown with relative fluorescence units (RFU) plotted against cycle number. The default setting of 10 times the standard deviation of fluorescence in all wells over the baseline cycles was used to calculate the threshold cycle or Ct value for a positive reaction (horizontal line). B: Standard curve analysis of the RNA amplification plots with Ct values plotted against starting copy numbers. Typical amplification plot derived from serial tenfold dilutions of recombinant plasmid pMD19-T-VP1 ranged from 10⁶ to 10¹ copies/ml (R² = 0.9993). C: Specificity analysis of the Real time RT-PCR was performed by using the genomic nucleic acid from poliovirus typeI-III; coxsackievirus A16, B1, B5, B3; human enterovirus 71; echovirus 6, 30; rotavirus A; sapovirus; norovirusII; astrovirus and enteral adenovirus strains as control. For the primer–probe sets, there were no positive results obtained except the RNA transcripts of the CVA6 virus. D: Clinical samples were identified by the real-time RT-PCR. By using the recombinant plasmid pMD19-T-VP1 as control, RNA transcripts from eight clinical samples give a typical amplification plot.