Development of a nine-variant reference material panel to standardize cell-free DNA detection

Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2024-07, Vol.416 (18), p.4123-4130
Main Authors: Niu, Chunyan, Zhang, Jiejie, Fang, Yan, Wang, Xia, Tang, Yanru, Dong, Lianhua
Format: Article
Language:English
Subjects:
Analytical Chemistry
Assaying
Biochemistry
Biomarkers
Biopsy
Characterization and Evaluation of Materials
Chemistry
Chemistry and Materials Science
Deoxyribonucleic acid
DNA
Food Science
Gene frequency
Laboratory Medicine
Monitoring/Environmental Analysis
Mutation
Mutation hot spots
Performance evaluation
Point mutation
Quality control
Reference materials
Research Paper
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Summary:Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.
ISSN:1618-2642
1618-2650
1618-2650
DOI:10.1007/s00216-024-05336-3