Multiplexed quantification of four neuroblastoma DNA targets in a single droplet digital PCR reaction.


  • C. Peitz
  • A. Sprüssel
  • R.B. Linke
  • K. Astrahantseff
  • M. Grimaldi
  • K. Schmelz
  • J. Toedling
  • J.H. Schulte
  • M. Fischer
  • C. Messerschmidt
  • D. Beule
  • U. Keilholz
  • A. Eggert
  • H.E. Deubzer
  • M. Lodrini


  • Journal of Molecular Diagnostics


  • J Mol Diagn 22 (11): 1309-1323


  • Detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive liquid biopsy approach. Major challenges of cfDNA analysis purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a suitable technology to analyze low levels of cfDNA. We here report two quadruplexed ddPCR assay protocols that (i) reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and (ii) accurately estimate ALK(F1174L) (3522, C>A) and ALK(R1275Q) (3824, G>A) mutant allele fractions using cfDNA as input. We optimized separation of positive and negative droplets to detect two targets in each ddPCR fluorescence channel by adjusting probe and primer concentrations for each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those for the respective duplexed assays. We present two robust quadruplexed ddPCR protocols applicable in the routine clinical setting to assess MYCN and ALK oncogene status that require only minimal plasma volumes.