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Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
Elmo W I Neuberger1, Alexandra Brahmer1, Tobias Ehlert1
1Department of Sports Medicine, Rehabilitation and Disease Prevention, University of Mainz, Albert-Schweitzer Str. 22, 55128, Mainz, Germany.
Scientific Reports
|July 1, 2021
Summary
Directly quantifying cell-free DNA (cfDNA) in plasma without isolation offers a cost-efficient method. Validated assays enable reliable cfDNA measurement, crucial for disease screening and understanding exercise impact in systemic lupus erythematosus (SLE) patients.
Area of Science:
- Biochemistry
- Molecular Biology
- Clinical Diagnostics
Background:
- Circulating cell-free DNA (cfDNA) shows promise for disease screening.
- DNA isolation is costly and time-consuming.
- Direct quantification PCR assays require rigorous validation for clinical use.
Purpose of the Study:
- To provide a comprehensive procedure for validating direct cfDNA quantification assays from blood plasma without DNA isolation.
- To validate 90 bp and 222 bp assays for studying cfDNA kinetics post-exercise in systemic lupus erythematosus (SLE) patients.
- To assess the impact of pre-analytical factors on cfDNA concentration.
Main Methods:
- Validation of direct quantification PCR assays for cfDNA according to ISO and bioanalytical guidelines.
- Assay validation included limit of quantification (LOQ), repeatability, and intermediate precision.
- Incurred sample reanalysis and pre-analytical factor assessment (centrifugation, temperature).
Main Results:
- Validated assays demonstrated ultra-low LOQ (0.47 and 0.69 ng/ml).
- Repeatability and intermediate precision were ≤11.6% and ≤12.1%, respectively.
- cfDNA increased twofold after exercise in SLE patients, normalizing within 60 minutes of rest; centrifugation and temperature did not affect cfDNA levels.
Conclusions:
- Established direct quantification assays provide reliable and cost-efficient measurement of cfDNA in small plasma volumes.
- The validated assays are suitable for clinical settings and can assess pre-analytical factor impacts.
- Demonstrated cfDNA kinetics post-exercise in SLE patients.
