3.2. Specificity and reproducibility of the single-tube duplex RT-PCR
Due to the complexity of clinical samples, specificity is an important indicator for evaluating and establishing detection approaches. In order to evaluate the specificity of the established single-tube duplex RT-PCR, this study used single-tube duplex RT-PCR to detect the M/N ratio of one MNA NB cell line (SK-N-BE2), two non-MNA NB cell line (SK-N-AS, SH-SY5Y) and one human umbilical vein endothelial cell (HUVEC), indicating that the M/N ratio of SK-N-BE2 is higher than SK-N-AS, SH-SY5Y and HUVEC. The M/N ratio of SK-N-AS, SH-SY5Y and HUVEC is less than 2 (Figure 2A). Furthermore, the amplification products ofMYCN and NAGK in NB tissues were sequenced based on the single-tube double RT-PCR method and the sequencing results were more than 99% similar to the target sequences (Figure 2B and Figure 2C). Therefore, the single-tube double RT-PCR method established in this study has high specificity in real sample analysis. In addition, the reproducibility of the developed single-tube duplex RT-PCR recipe was investigated by five consecutive assays where the RSDs for MYCNand NAGK were determined to be 1.3% and 0.7%, respectively (Figure S3). The results show that the developed single-tube duplex RT-PCR recipe has acceptable reproducibility. In summary, the developed approach has high specificity in real sample analysis and acceptable reproducibility.