Digital PCR: a reference measurement procedure for infectious disease molecular testing

Abstract

Metrology facilitates the advancement of accurate, SI-traceable measurements. In many cases for molecular measurements, there is a lack of standardization of methods across laboratories and platforms which leads to variability in results. Standardizing methods may promote accuracy and reproducibility and facilitate data comparison across studies and platforms. Polymerase chain reaction (PCR) based techniques represent an ideal first diagnostic solution for infectious pathogens and are deployed to detect the genomes of infectious agents (DNA/RNA) as they can be quickly designed and demonstrate high sensitivity and specificity. The COVID-19 pandemic demonstrated how diagnostic testing was crucial in tracking and diagnosing patients infected with SARS-CoV-2, but initial testing was largely unstandardized and it is possible that patients were incorrectly diagnosed as a result. Digital PCR (dPCR) is a single molecule enumeration technique that can reproducibly quantify nucleic acid sequences without using calibration, offering a route to provide reference values to support PCR testing and diagnostics. dPCR has been used for quantitative detection of pathogens and has been established to serve as a potential reference measurement procedure (RMP) for the accurate quantification of nucleic acids from pathogens such as Mycobacterium tuberculosis, human cytomegalovirus, human immunodeficiency virus-1, some of which are already listed as reference methods in the JCTLM database. In the recent study, National Metrology Institutes/Designated Institutes (NMIs/DIs) developed a potential reverse transcription-dPCR (RT-dPCR)-based RMP for SARS-CoV-2 RNA. This approach was deployed to support external quality assurance (EQA) by assigning metrologically traceable values to whole viral SARS-CoV-2 EQA material. The value assignment standardized the SARS-CoV-2 RNA concentration and was used to determine the limit of detection. This collaboration has led to the support of international standardization of molecular testing using dPCR as an RMP, thereby ensuring accurate and reproducible nucleic acid reference values for the EQA materials. Current activities include participation in the CCQM Nucleic Acid Working Group (NAWG) for international interlaboratory comparison studies (pilot and key comparisons) to support the development of SI-traceable dPCR based RMPs. This poster will describe some of the recent work on dPCR-based RMPs and discuss how this can be applied to improve nucleic acid analysis measurements on a global scale in EQA schemes, clinical laboratories and harmonization studies for infectious disease diagnostics.