Accurate, comparable viral load determination is essential for clinical diagnostics and treatment monitoring. However, there is no direct primary method to count viral particles. Laboratories typi-cally measure surrogate quantities, such as viral DNA or protein, which prevents direct data comparison and traceability to the International System of Units (SI). As different analytical approaches quantify distinct molecular components rather than the same physical entity, their results inherently reflect different measurands and therefore cannot be directly compared.
To address this, this thesis proposes a method traceable to the International System of Units (SI) to estimate viral load, using Human Cytomegalovirus (HCMV) as a model. It combines two primary reference measurement procedures: droplet digital PCR (ddPCR), a technique for the absolute counting of viral DNA molecules, and isotope dilution mass spectrometry (IDMS), a method for precise measurement of specific proteins. Together, these methods enable metrologically accurate estimation of viral particle numbers by linking measurements of viral genetic material and proteins through the fixed ratio (stoichiometry) of components in the viral capsid (the protein shell enclosing the genetic material).
Building on this approach, human cytomegalovirus (HCMV) was produced in a cell culture, and viral particles were purified by density gradient ultracentrifugation, a technique that separates components based on their density. Proteomics analysis, an assessment of all proteins within a sample, then identified the major capsid protein (MCP) and triplex subunits Tri1 and Tri2 which were present in a fixed stoichiometry enabling the quantification of the viral particle targets. Signature peptides (unique short protein fragments) from these proteins were selected and obtained commercially. Amino acid analysis with isotope dilution mass spectrometry, a precise method for measuring molecules using labelled standards, provided SI-traceable value assignment. A robust LC–MS/MS (liquid chromatography–tandem mass spectrometry) method in multiple-reaction-monitoring mode was developed for quantifying MCP, Tri1, and Tri2. In parallel, ddPCR (droplet digital polymerase chain reaction) assays targeting the UL54 gene determined absolute genome copy numbers without the need for calibration curves. Multiple extractions showed good reproducibility, with extraction efficiency as the main source of uncertainty.
To support standardisation, a plasmid-based laboratory standard (pYB01) containing the conserved human cytomegalovirus (HCMV) UL54 gene was generated, sequence-verified, and given a quantitative value using droplet digital PCR (ddPCR). This calibrator for quantitative PCR (qPCR) assays is accessible, reproducible, and traceable, enabling standardized viral genome quantification in routine laboratories.
IDMS-based quantification provided independent capsid protein results, complementing molecular measurements. These were converted into capsid numbers using established cryoelectron microscopy stoichiometries. Integrating the ddPCR- and IDMS-derived data yielded an estimated concentration of ~6 x 10⁷ virions per milligram. The uncertainty budgets of both methods overlapped, confirming the reliability of these independent primary approaches.
A detailed uncertainty analysis following the Guide to the Expression of Uncertainty in Measurement (GUM) identified DNA extraction efficiency and the protein-to-capsid conversion factor as the main sources of uncertainty.
Combining ddPCR and IDMS enables SI-traceable viral load measurement. This approach bridges metrology and virology, improving accuracy, reproducibility, and comparability across laboratories. It also supports certified reference materials and standard procedures, strengthening diagnostics and monitoring.