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Objavljeno v DiRROS: 03.04.2026; Ogledov: 277; Prenosov: 144
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Ključne besede: celične kulture, tumorji, molekularna diagnostika, ekstrakcijske metode, kontrola kakovosti, GenomeMET
Objavljeno v DiRROS: 11.03.2026; Ogledov: 346; Prenosov: 113
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Povzetek: The genotyping workflow described uses digital PCR (dPCR) to detect and quantify drug resistance mutations in human cytomegalovirus (HCMV). The method focuses on the detection and quantification of three common mutations in the UL97 gene at codons 460, 594, and 595, which are responsible for the majority of ganciclovir-resistant clinical isolates. The dPCR approach offers high sensitivity and accuracy, making it suitable for routine testing as well as a reference measurement procedure for external quality assessment schemes. The workflow includes several key steps: DNA isolation, preparation of the dPCR reaction mixture, partitioning, thermocycling, and data analysis. This method improves the detection capabilities of HCMV drug resistance and provides a robust and efficient tool for clinical and research applications.
Ključne besede: digital PCR, human cytomegalovirus, antimicrobial drug resistance, mutations
Objavljeno v DiRROS: 25.09.2025; Ogledov: 566; Prenosov: 118
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Povzetek: 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 (RTqPCR) 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.
Ključne besede: nucleic acid amplification tests, dPCR, reference measurement procedure, external quality assessment, SARS-CoV-2
Objavljeno v DiRROS: 14.04.2025; Ogledov: 1266; Prenosov: 909
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Povzetek: Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals (‘measurement uncertainties’) were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.
Ključne besede: RNA copy number ratio, RT-qPCR, gene expression, normalisation, standardisation, molecular diagnostic, transcriptomics, cancer, diagnostics, biomarker identification and validation
Objavljeno v DiRROS: 04.03.2025; Ogledov: 1047; Prenosov: 569
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