Dual isothermal amplification all-in-one approach for rapid and highly sensitive quantification of plasma circulating MYCN gene of neuroblastoma

Abstract

A dual isothermal amplification assay with dual fluorescence signal detection strategy, named dual isothermal amplification all-in-one approach, was developed for rapid, one-step, highly sensitive quantification of plasma circulating MYCN copy number of neuroblastoma (NB). The developed strategy consisted of rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) on a real-time PCR system using highly specific probe, molecular beacon (MB), as detection probe. The developed strategy possessing a broad linear dynamic range of 10 aM to 1 pM for both target gene (MYCN) and reference gene (NAGK). The ratio of the MYCN copy number to NAGK copy number (M/N ratio) was detected by the developed approach in cell lines, NB tumor tissues, hepatoblastoma tumor tissues and Wilms' tumor tissues, to which the M/N ratios were consistent with previous reports. In particular, the M/N ratio in NB clinical tissue specimens and NB plasma specimens detected with the developed approach were in keeping with the standard RT-PCR approach. More importantly, the M/N ratio in NB tissue samples and corresponding plasma samples of NB patients were consistent with each other with a correlation coefficient of 0.9690, indicating that plasma circulating MYCN is a promising indicator for the risk classification of NB.

Keywords: All-in-one; LAMP; MYCN; Molecular beacon; Neuroblastoma; RCA.