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.