Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease

Huggett, Jim F.; O'Sullivan, Denise M.; Cowen, Simon; Cleveland, Megan H.; Davies, Kerrie; Harris, Kathryn; Moran-Gilad, Jacob; Winter, Amanda; Braybrook, Julian; Messenger, Michael

doi:10.1016/j.mam.2024.101275

Molecular Aspects of Medicine

2024

DOI: 10.1016/j.mam.2024.101275

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Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease

Jim F. Huggett,

Denise M. O'Sullivan,

Simon Cowen

et al.

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Cited by 4 publications

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Advancements of prokaryotic Argonautes in molecular diagnostics and future perspectives

Shang,

Gao,

Wei

et al. 2025

TrAC Trends in Analytical Chemistry

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Advancements of prokaryotic Argonautes in molecular diagnostics and future perspectives

Shang,

Gao,

Wei

et al. 2025

TrAC Trends in Analytical Chemistry

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The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing

Falak,

O’Sullivan,

Cleveland

et al. 2024

Clinical Chemistry

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Background Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available. This study investigated digital PCR (dPCR) RMP delivery of traceability for NAAT external quality assessment (EQA). Methods Three National Metrology Institutes (NMIs) applied reverse transcription (RT)-dPCR as a candidate RMP to estimate the RNA quantity in 32 independent severe acute respiratory syndrome coronavirus 2 materials. The results were combined to value assign the respective materials: 21 materials were used in 6 rounds of EQA over 17 months for 61 laboratories for COVID-19 testing results compared with reference values. Results The agreement between the 3 NMIs showed <2-fold difference between laboratories. EQA laboratory reverse transcription quantitative PCR (RT-qPCR) values estimation of viral RNA quantity showed good median agreement with RT-dPCR reference value; however, RT-qPCR differences were generally between 10- and 50-fold between laboratories. Conclusion This work demonstrates how RT-dPCR can provide reference values for whole virus materials for NAAT quality assurance. RT-dPCR values guided EQA control material selection and provided EQA participants with traceability to RNA copy number delivered through the RMP. This approach can be used to support routine reference material use as well as to standardize quality assurance for NAATs where established reference materials are not available, such as in disease outbreaks.

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A duplex HiFi-LAMP assay for screening of two novel human circoviruses HCirV-1 and HCirV-2

Xu,

Zhang,

Chen

et al. 2024

BMC Infect Dis

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Circoviruses belong to the family Circoviridae, which is classified into two genera, Circovirus and Cycloviru s. Some circoviruses have been identified in various organisms and/or their fecal samples and might be associated with diseases in their hosts. However, few circoviruses are reported in human. Recently, two novel circoviruses HCirV-1 and HCirV-2 were identified in humans through next-generation sequencing and were defined as the species of Circovirus human in the family Circoviridae . Both viruses are suspected to be associated with liver diseases, particularly among immunosuppressed people, showing potential health implication. Investigation on the prevalence of both viruses, and identification of vulnerable population are important and need a rapid, accurate, and specific assay for detecting and screening the two viruses. In this study, we developed a duplex HiFi-LAMP assay for simultaneous detection of HCirV-1 and HCirV-2. The assay exhibits high sensitivity with LOD of 64 and 49 copies per 25 µL reaction for HCirV-1 and HCirV-2, respectively. The duplex assay was demonstrated to have a rapid reaction time within 35 min. Clinical screening tests showed that neither HCirV-1 nor HCirV-2 were detected among 875 patients with infection of HBV, HIV-1 or other viruses. Large-scale screening of both viruses in diverse populations is encouraged to enhance our understanding of their relevance to various diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-024-10283-6.

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Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease

Cited by 4 publications

References 33 publications

Advancements of prokaryotic Argonautes in molecular diagnostics and future perspectives

Advancements of prokaryotic Argonautes in molecular diagnostics and future perspectives

The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing

A duplex HiFi-LAMP assay for screening of two novel human circoviruses HCirV-1 and HCirV-2

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