3.1. Feasibility and sensitivity of the single-tube duplex RT-PCR
The feasibility of single-tube duplex RT-PCR, using two-color MB (MYCN MB and NAGK MB) to detect MYCN andNAGK in one PCR tube, was systematically studied. First, to evaluate the feasibility of the MYCN MB and NAGK MB in quantifying its target sequence, the features of MBs were studied. The secondary structure of MYCN MB and NAGK MB were predicted by Quikfold Fast Folding (http://www.unafold.org/Dinamelt/applications/quickfold.php), which both possessed a stem-loop structure (Figure S1A and Figure S1B). TheMYCN MB and NAGK MB were modified at its 5’ end with a fluorophore (FAM and VIC, respectively) and at its 3’ end with a quencher (BHQ-1). The fluorescence values of MYCN MB andNAGK MB were detected in the presence and absence of the target sequence, and the optimal reaction temperature of MYCN MB andNAGK MB were both 28 °C (Figure 1A and Figure 1B). The fold change value is the value of the fluorescence of MB in the presence and absence of target sequence. The optimum reaction temperature for MYCN MB and NAGK MB were both 28 °C (Figure 1A and Figure 1B). We then investigated the sensitivity of MB to detect target sequences. When the concentration ofMYCN MB was 1000 nM, the MB fluorescence signal was linearly related to the target concentration (with a range from 10 to 1000 nM). The correlation equation was y = 25.735 x + 2250.8 (R2 = 0.9976), where, y is the fluorescence. The fold change value is the value of the fluorescence of MB in the presence and absence of target sequence of MYCN MB and xis the concentration of target sequence (Figure 1C). Meanwhile,NAGK MB fluorescence signal is linearly related to target concentration. The correlation equation was y = 45.981 x + 3016.4 (R2 = 0.9938) where y is the fluorescence value of NAGK MB, x is the concentration of target sequence (Figure 1D). Those results demonstrated that MYCNMB and NAGK MB have high sensitivity to detect the target sequence. In conclusion, the above experimental results demonstrated the feasibility of the designed MB to detect target sequences.
Sensitivity and specificity are an important factor in evaluating the developed approaches due to the lower content of MYCN in plasma and tissue. To test whether a single-tube duplex RT-PCR method could effectively detect MYCN and NAGK quantitatively, experiments were carried out with the results depicted in Figure 1E and F. Figure 1E shows the real-time fluorescence curves of differentMYCN copy numbers where the C T values increase with a decrease in MYCN copy numbers. On the logarithmic scale, C T values and MYCN copy numbers yielded a good linear relationship on 7 orders of magnitude from 10 to 107 copies. The correlation equation is y = −3.3882 lg x + 36.583 (y and x are theC T value and NAGK copy number, respectively) and the correlation coefficient is R2 = 0.9970. Figure 1F shows the real-time fluorescence curve of differentNAGK copy numbers where the C T value increases with a decrease in NAGK copy number. On the logarithmic scale, C T values and NAGK copy numbers yielded a good linear relationship on 7 orders of magnitude from 10 to 107 copies. The correlation equation is y = −3.473 lg x + 36.56 (y and x are theC T value and NAGK copy number, respectively) and the correlation coefficient is R2 = 0.9967. Therefore, the developed single-tube duplex RT-PCR can quantitatively detect MYCN and NAGK in a single tube with high sensitivity.