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

Addressing variability in rapid antigen detection kits with viral protein reference materials

Lateral flow assay (LFA) rapid antigen detection tests (RADT) became a widely used diagnostic tool during the COVID-19 pandemic, and they remain an indispensable strategy to identify infected individuals and prevent transmission of the SARS-CoV-2 virus in vulnerable populations. Many hundreds of these COVID 19 RADTs have been developed worldwide and are commercially available, yet a lack of relevant comparative analyses required to inform on absolute analytical sensitivity and performance has limited end-user ability to accurately compare brands for decision making. Achieving comparability between manufacturers presents a significant metrological challenge yet is indispensable to both effective regulation and consumer confidence. The analytical sensitivity, or limit of detection, of COVID-19 RADTs is most often reported as the median tissue culture infectious dose (TCID50) of intact viral culture. However, the vast majority of test kits detect only the viral nucleocapsid protein (Np). As viral cultures are notoriously difficult to standardize and can be challenging to ship across international borders during a pandemic, we developed two Np reference materials (RM) – a fluorescent-labelled Np fusion protein (NP-GFP) using bacterial expression, and an unlabeled protein (NCAP-1) produced in mammalian cells. The protein contents of the RMs were assigned using isotope dilution mass spectrometry and/or spectroscopic detection. Protein higher-order structure (HOS) can affect how efficiently LFA detection antibodies recognize their antigen. Protein HOS was interrogated via size-exclusion chromatography, which revealed that NP-GFP existed exclusively as a tetramer, whereas NCAP-1 consisted of a mixture of tetramers and hexamers. We then assessed the analytical sensitivities of 26 commercially available RADTs using a dilution series of both protein RMs. Despite the differences in expression system, oligomeric state, and buffer composition, both RMs generated highly comparable results within each RADT. However, we demonstrate orders of magnitude differences in analytical sensitivities among distinct RADTs, and find little correlation with the TCID50 assay values reported by manufacturers. These results indicate that consumers’ choice of test kit can potentially influence their perceived infection status. The emergence of another viral pandemic would undoubtedly result in the production and public marketing of a plethora of new antigen detection test kits. Our data highlight the need for, and practicality of, readily available protein RMs to insure batch-to-batch quality and accuracy of RADTs across manufacturers. Development and regulation of the requisite antigen protein RMs thus represents a crucial component in a pandemic preparedness strategy.

Advancement of LAMP technologies for rapid and accurate diagnosis of infectious diseases

SUMMARY Loop-mediated isothermal amplification (LAMP) is a type of isothermal nucleic acid amplification test (NAAT). Compared to more traditional NAATs, like polymerase chain reaction (PCR), LAMP is run at one consistent temperature, utilizes four to six primers, and uses Bst polymerase. Some advantages that are provided by these differences are that LAMP has lower equipment requirements, resiliency to common PCR inhibitors, and a rapid time to result. Unfortunately, some disadvantages are also present due to these differences, such as more challenging primer design, reduced multiplexing capability, and difficulty with quantitation. Due to its unique characteristics, LAMP has proven to be an attractive option for molecular diagnostics in resource-limited, point-of-care, and near-patient testing settings. However, its adoption in clinical settings remains limited compared to its widespread use in research. In this review, we cover various detection methods within LAMP, recent advancements including extraction, multiplexing, and quantification, as well as key clinical applications of LAMP from a syndromic perspective. We also explore the commercial potential of LAMP and how it can be leveraged in resource-constrained settings and clinical laboratories, especially where early, affordable, and rapid diagnosis is essential.

Best Practice of Biometrology to Support Nucleic Acids Detection of Infection Disease

In response to the demand for metrology in the context of the SARS-CoV-2 epidemic, National Institute of Metrology (NIM), China has undertaken a series of initiatives in the field of infectious disease testing, ranging from cutting-edge research to practical application. Three reference measurement procedures (RMP) published at JCTLM specify the operating specifications for SARS-CoV-2 (open reading frame 1ab, ORF1ab and nucleocapsid protein, N and Envelope, E gene) RNA copy number concentration by reverse transcription digital PCR (RT-dPCR). Certified Reference Materials including in vitro transcribed RNA, genomic RNA and Pseudoviral RNA of SARS-CoV-2 and different variants (GBW0928 to GBW09318) have been developed and widely applied for practical application. These reference materials were widely used by more than 1000 institutes or companies, including IVD manufacturers, CDCs, clinical laboratories, universities and institutes. Especially in helping many IVD manufacturers obtain certification for their testing kits from National Medical Products Administration of China (NMPA), FDA and CE, it has played an important role in providing metrological traceability. To investigate the international comparability in nucleic acid measurement and supporting CMC claim, NIM successfully coordinated two rounds’ comparisons for SARS-CoV-2 (CCQM-P199B and CCQM-K181) with LGC, NIBSC and NIST. Furthermore, to support other NMIs in APMP region to establish the capability in measurement of SARS-CoV-2, an international training course of “Accurate Measurement of SARS-CoV-2 and Development of Nucleic Acids Reference Material” was organized by NIM incorporated with HSA, KRSISS and NIST. Additionally, three rounds of proficiency testing for SARS-CoV-2 RNA and its different variants were conducted to enhance the capacity and guarantee the consistency of testing facilities in China. In 2023-2024, NIM has provided invaluable assistance to China CDC in the ongoing monitoring of SARS-CoV-2, particularly in the detection of the virus in urban sewage. This support includes providing simulated samples of Pseudoviral virus of SARS-CoV-2, as well as enriching the virus in urban sewage and employing highly accurate measurement methods. The whole framework can be used as the best practice of biometrology to support nucleic acids detection of infection disease and get prepared for future epidemic.

New candidate reference measurement procedure for NRAS p.Q61R quantification by digital PCR.

Accurate and comparable quantification of somatic mutations is essential for precision oncology, as clinical decision-making increasingly relies on the quantification of molecular biomarkers. Despite major technological advances, inter-laboratory variability and the lack of metrological traceability remain significant barriers to harmonization and confidence in mutation testing results. Reference Measurement Procedures (RMPs) represent a critical framework to address these challenges by anchoring molecular measurements to common quantitative standards. Here, we describe the development and validation of a candidate RMP for the detection and quantification of the clinically relevant NRAS p.Q61R mutation using digital PCR (dPCR). The assay was systematically optimized to maximize specificity and minimize cross-reactivity between wild-type and mutant alleles. Analytical characterization demonstrated excellent linearity across a broad range of variant allele frequencies (vAF), with a limit of detection of 0.1 %. Precision studies performed on commercially available circulating tumour DNA reference materials (RM) showed good repeatability and intermediate precision, while a full measurement uncertainty budget confirmed the robustness of the approach. Comparison with a commercial dPCR assay provided independent support for assay comparability and consistent vAF estimates across the investigated range. Preliminary inter-laboratory assessment supported transferability of the candidate RMP and comparability of the resulting measurements. Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification. Its implementation can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.

PCR and molecular diagnostics: Biotechnologic integration in clinical laboratories.

A new generation of molecular tests has revolutionized laboratory medicine through allowing for quick, sensitive, and precise identification of the pathogen; genetic mutation or alteration; and disease-related bio-markers. Of all the molecular techniques that exist today, PCR is still the most versatile, and clinically applicable technique available. This paper reviews the development of PCR technology from traditional amplification to advanced platforms including qPCR, dPCR, RT-PCR, and multiplex PCR. Each of the advanced platforms reviewed will be examined for their contributions to both the accurate detection, and quantification of nucleic acids along with the ability to perform high throughput analyses. Additionally, this paper also discusses how PCR technology can be integrated with additional technologies such as NGS, CRISPR-based diagnostic systems, microarrays, and epigenetic profiling to provide a larger view of disease states than was previously possible. Specifically, the focus of this paper will include many clinically relevant biomarkers and molecular targets used in cancer research including EGFR, KRAS, BRAF, BCR-ABL1, circulating tumor DNA (ctDNA), and methylated SEPT9. These biomarkers and molecular targets have greatly enhanced early diagnosis, prognostication, treatment monitoring and precision medicine in cancer research and other areas. The use of PCR-based technologies in diagnosing infectious diseases, tracking AMR, detecting heritable disorders, studying microbial communities, and MRD monitoring were also discussed. Further discussion included current trends in integrating multiple omics data types into a single platform using AI assisted data analysis tools. Liquid biopsy technologies and point-of-care molecular test platforms were also discussed. Although there are continuing challenges related to costs associated with developing these technologies, establishing standardized protocols for each technology type developed, having skilled technicians develop and validate these technologies, and managing large amounts of data generated when using these technologies; continued advances are providing greater access to these technologies to clinicians and ultimately patients.

A point-of-care testing platform for pathogen nucleic acid analysis utilizing paper-based extraction and portable qPCR.