Related Experiment Video
Updated: Aug 28, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Development and Broad Application of a Double Drop-Off ddPCR Assay for Simultaneous Detection of Four FGFR3 Mutations
Eleni Thanou1, Nikos Gavalas2, Eleni Kabrani3
1Analysis of Circulating Tumor Cells, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece.
Background:
Fibroblast Growth Factor Receptor 3 (FGFR3) mutations are common and clinically relevant alterations in bladder cancer, with implications for diagnosis, monitoring, and patient selection for targeted therapies. Tissue-based testing is often limited by sample availability, invasiveness, and the need for repeated sampling. This study aimed to develop and analytically validate a sensitive double drop-off droplet digital polymerase chain reaction (ddPCR) assay for simultaneous detection of four FGFR3 hotspot mutations in tissue and liquid biopsy samples.
Methods:
The assay targeted four FGFR3 mutations (S249C, R248C, Y373C, G370C) using reference probes that generate a constant fluorescence signal and wild-type-specific drop-off probes that lose binding when a mutation is present, thereby distinguishing wild-type double-positive droplets from mutant droplets with reduced drop-off fluorescence. Analytical validation was performed using synthetic mutant oligonucleotides, wild-type genomic DNA, and cell-free DNA (cfDNA) from healthy donors (HDs). Specificity, limit of blank (LOB), limit of detection (LOD), and assay precision were evaluated. Performance was compared with next-generation sequencing (NGS) in formalin-fixed paraffin-embedded (FFPE) tissue DNA. FGFR3 mutations were also assessed in matched plasma and urinary cfDNA from bladder cancer patients.
Results:
The assay demonstrated clear cluster separation, no cross-reactivity, and reliable detection of all mutations down to 0.2% mutant allele frequency (MAF). Strong agreement was observed with a mutation-specific singleplex ddPCR assay for S249C. Concordance with NGS in tissue DNA was 74.2%, with ddPCR identifying additional low-abundance mutations not reported by NGS. FGFR3 mutations were detected in plasma and urinary cfDNA, with complete concordance between matched plasma and urine samples.
Conclusions:
This ddPCR assay provides a rapid, sensitive, and cost-effective method for detecting clinically relevant FGFR3 mutations and may complement sequencing-based approaches for molecular monitoring using tissue, plasma, and urine specimens.
