Well-characterized reference materials are essential for ensuring harmonization and accuracy in nucleic acid-based tests, such as quantitative PCR (qPCR). Digital PCR (dPCR) is particularly advantageous for measuring the absolute concentration of specific nucleic acid sequences within a complex background of non-target sequences. This capability makes dPCR ideal for characterizing nucleic acid-based reference materials. National Metrology Institutes (NMIs) are increasingly adopting dPCR for certifying reference materials due to its superiority over indirect methods like UV-spectroscopy. However, dPCR requires careful optimization and comes with certain limitations and considerations.
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Fig. 1 Digital PCR measurements throughout the reference material process (Cleveland M. H., et al. 2024).
How Digital PCR Works
Digital PCR (dPCR) shares the same basic chemistry and thermal cycling parameters as quantitative PCR (qPCR), aiming to amplify a specific nucleic acid sequence using primers. The amplification is measured with fluorescent probes or non-specific DNA binding dyes. However, dPCR fractionates the reaction into hundreds or thousands of low-volume partitions, either as droplets in an oil emulsion or fixed chambers within a cartridge. Some partitions will contain a target molecule, while others will not. By knowing the partition volume, the initial concentration of the target sequence can be calculated using Poisson statistics, correcting for partitions that might contain more than one target molecule.
Unlike qPCR, dPCR does not require an external calibrant for absolute quantification. It uses an end-point measurement, counting positive and negative partitions after all PCR cycles. Some dPCR instruments also offer real-time measurements, tracking fluorescence in each partition per cycle to create amplification curves.
Reference Materials
The Importance of Reference Materials
Reference materials are defined by their homogeneity and stability, ensuring they are suitable for use in measurement processes or examinations. They calibrate measurement systems, facilitate quality control, and allow performance assessments of instruments and methods. Certified reference materials (CRMs) provide certified quantities, associated uncertainties, and metrological traceability. CRMs are vital for ensuring consistent results across different laboratories and over time.
Nucleic Acid-Based Reference Materials
Nucleic acid analysis is crucial in various fields such as clinical diagnostics, food analysis, and environmental monitoring. With the increasing application of nucleic acid-based tests, there is a growing need for reference materials to develop, validate, and calibrate these tests. Reference materials are necessary for both qualitative and quantitative analysis. For instance, the National Institute of Standards and Technology (NIST) has developed genomic DNA standards for measuring gene amplifications relevant to cancer treatment. Similarly, synthetic partial SARS-CoV-2 RNA has been used to calibrate qPCR measurements of SARS-CoV-2 in wastewater samples.
Historical Measurement of Nucleic Acid-Based Reference Materials
Before the routine use of dPCR, NMIs, and other producers used indirect methods such as UV spectroscopy and nucleic acid binding dyes to certify nucleic acid concentrations. These methods, however, measured total nucleic acid content and could not provide information on sequence identity or fragment integrity. Indirect methods also required extraction processes that could result in unknown losses of nucleic acid. In contrast, dPCR specifically measures target sequences, making it more accurate and less prone to contamination effects.
Factors Affecting the Accuracy of dPCR Measurements
dPCR is replacing indirect methods for certifying nucleic acid-based reference materials due to its ability to measure low concentrations of target sequences without external calibrators. Factors affecting dPCR measurements include secondary structure and degradation, which also affect qPCR. Therefore, dPCR-certified reference materials are more commutable with clinical specimens measured using qPCR methods. Despite its advantages, dPCR requires optimization and parallel use of other quantification methods to ensure accuracy.
Assay Design
Using multiple well-validated dPCR assays that span the target sequence helps to avoid underestimation due to suboptimal assay design or secondary structure. Assays should be optimized for parameters like annealing temperature, primer/probe concentration, and cycle number. For RNA measurement using RT-dPCR, the choice of primers for reverse transcription is crucial for efficiency.
Selection of Appropriate Reference Genes for Ratio-Based Materials
Ratio-based measurements, such as those used in cancer testing and prenatal screenings, require thoroughly characterized reference genes. Multiple reference genes should be compared to ensure accurate measurements. For example, in HER2 gene copy number certification, NIST used four unique reference genes to calculate HER2 ratios across different cell lines.
Partition Volume
Accurate calculation of starting concentration depends on precise partition volume measurement. While manufacturers provide nominal partition volumes, slight variations can occur. NMIs may measure partition volumes for SI-traceable measurements, but in some dPCR platforms, user measurement is infeasible, requiring reliance on manufacturer-provided values.
Subsampling Error for Rare Event Quantification
Subsampling error occurs when only a portion of a larger sample is tested, especially for low-concentration targets. The distribution of results often follows a Poisson distribution rather than a Gaussian distribution. For rare event quantification, subsampling introduces significant uncertainties.
Preparation of the Nucleic Acid Material
The secondary structure and integrity of nucleic acid affect dPCR measurement accuracy. Circular DNA materials should be linearized, and DNA fragmentation may be necessary to improve accuracy. Enzyme digestion or sonication can reduce viscosity and separate tandem gene copies, but these treatments should be considered on a case-by-case basis.
Other Preanalytical Factors
DNA extraction methods can impact dPCR quantification. Inhibitors in the extracted DNA solution can interfere with partitioning and subsequent analysis. The state of DNA (double-stranded or single-stranded) should be considered during extraction, transportation, and storage.
Additional Sources of Uncertainty with dPCR Measurements
Uncertainties in dPCR measurements also arise from factors like partition classification and Poisson distribution effects. Manual threshold setting can be subjective, and automated programs may help reduce variability. False positive rates and assay design also contribute to measurement uncertainty.
Limitations of dPCR for Reference Material Characterization
Materials Requiring Extraction are More Difficult to Fully Characterize
While dPCR can be SI-traceable for purified DNA and RNA solutions, characterizing materials that require nucleic acid extraction is challenging. Extraction methods vary in efficiency and can introduce bias. Direct dPCR methods, involving only a heat lysis step, may still face inhibition issues.
Uncertainty About Reverse Transcription Efficiency for RNA Materials
Reverse transcription efficiency affects RNA quantification. One-step RT-dPCR may underestimate RNA concentration due to less than 100% efficiency, while two-step RT-dPCR may overestimate it. Comparisons with orthogonal methods are necessary to understand and improve reverse transcription efficiency.
Inability to Amplify Highly Fragmented Materials
Highly fragmented nucleic acids, like cell-free DNA (cfDNA) in liquid biopsies, pose challenges for dPCR. The detection efficiency of cfDNA is higher with shorter amplicons, but quantifying highly fragmented ultrashort cfDNA remains difficult. Accurate quantification in liquid biopsies requires considering amplicon length and fragment length dependencies.
Conclusion
dPCR offers significant advantages for the characterization of nucleic acid-based reference materials, providing absolute quantification of specific sequences without the need for external calibrators. Its precision and accuracy make it increasingly favored by National Metrology Institutes for certified reference materials. However, optimizing dPCR assays, considering preanalytical factors, and addressing inherent limitations are crucial for ensuring reliable measurements. As dPCR technology advances and more orthogonal methods are employed, the accuracy and utility of nucleic acid-based reference materials will continue to improve, supporting diverse applications in clinical diagnostics, food analysis, environmental monitoring, and beyond.
Reference
- Cleveland M. H., et al. Digital PCR for the characterization of reference materials. Molecular Aspects of Medicine. 2024, 96: 101256.
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