Abstract 2930: Intra- and inter-run repeatability assessment of the naica® 6-color digital PCR system for multiplex genetic detection
Authors: Fatien Jean , Foret Benjamin , Gourdy Oriane , Mallory Allison C. 
Published: June 2022

The genomic analysis of clinical samples, whether it be frozen tissue, FFPE or liquid biopsies, requires the ability to quantify low-level genetic aberrations in a complex DNA background with high precision and sensitivity. Stilla Technologies’ 6-color naica® system is an ultrasensitive, easy-to-use digital PCR technology capable of quantifying multiple biomarkers in a single reaction.

The naica system workflow is comprised of optimized master mix reagents, microfluidic consumables, a 2-in-1 droplet generator and thermocycler, and a 6-color imager. For data analysis, the Crystal Miner analysis software allows for flexible sample analysis and inciteful data visualization. Altogether, the naica system workflow requires only a single pipetting step, thereby considerably limiting the variability contributed by the operator. The naica multiplex PCR MIX ensures optimal simultaneous quantification of multiple targets across the five-log dynamic range of the naica system, as well as robust quantification of low target concentrations in a highly abundant and complex DNA background. R² scores &gt; 0.99 were obtained for all targets of a 6-color detection panel ranging from 0.2 to 13000 copies (cp)/µL.

As repeatability is a key performance criterion to evaluate the quality of a digital PCR system and detection assay, we also assessed the intra- and inter-run repeatability of the naica® system with a 6-color assay. Three target concentrations from three different DNA sources were tested at 200, 2000, and 10,000 cp/μL. To allow robust statistical analysis on the means of concentration and variance of concentration, three runs of a total of 36 wells were performed for each target concentration using Stilla Technologies’ highly sensitive Sapphire chip. Our results showed that at each of the three tested concentrations, the inter-run and intra-run variability of the 6-color naica® system was less than 6%. For each of the three tested concentrations, no significant inter-run or intra-run differences (p&lt;0.05, two-tailed Mann-Whitney statistical test) were found, neither on the concentration level nor on the variance of concentration level. The absence of a significant difference implies that among multiple runs neither the well position in the chip nor the chip position in the Geode or Prism6 instrument has a significant impact on the results. Hence, any sample, including positive and negative control samples, can be loaded anywhere on the chips without significantly impacting the results, confirming high performance while ensuring ultimate flexibility in the experiment design.

Citation Format: Jean Fatien, Benjamin Foret, Oriane Gourdy, Allison C. Mallory. Intra- and inter-run repeatability assessment of the naica® 6-color digital PCR system for multiplex genetic detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2930.