Biology subjects

Temisak, S.

Publications and source records attributed to Temisak, S..

3 recordsLinked to original sources

bioRxiv2024

CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 109 /mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 "HIV-1 RNA copy number quantification" (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene of in vitro transcribed RNA molecules at low ({approx} 103 /L) and high concentration ({approx} 109 /L) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) (n = 4) and orthogonal methods: single molecule flow cytometric counting (n = 2), high performance liquid chromatograph (HPLC) (n = 1) and isotype dilution-mass spectrometry (ID-MS) (n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different (p = 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs. 808 /L; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.

molecular biology↗

bioRxiv2023

Standardisation of cell-free DNA measurements: An International Study on Comparability of Low Concentration DNA Measurements using cancer variants

For the impact of genomic testing from liquid biopsies to be maximized, mechanisms to ensure reproducible and comparable test performance will be required. This can be established and maintained through reference measurement procedures and materials with property values that are internationally comparable through traceability to a common standard. To achieve this objective, an interlaboratory study was organised to explore digital PCR (dPCR) for standardisation of cell-free DNA (cfDNA) quantification. Blinded samples of wild-type/variant mixtures of two DNA sequences (BRAF p.V600E single nucleotide variant or EGFR exon 19 deletion) were provided to 12 laboratories. Laboratories independently designed and applied dPCR assays to determine absolute and relative quantities, with no guidance provided to harmonise the approach. The mean and coefficient of variation (CV) of copy number concentrations for variant sequences were 18 copies/L (CV 7.2%) (BRAF variant sample) and 9 copies/L (CV 25%) (EGFR variant sample) while the mean variant allele frequencies (vAF) were 8.0% (CV 5.3%) and 0.080% (CV 29%) respectively. This study demonstrated that dPCR was capable of exceptional technical accuracy for variant copy number concentration and vAF, even when different assays and platforms were used. This implies that dPCR offers a unique analytical methodology that can be deployed globally in supporting comparability for cfDNA testing based on the existing framework of the International System of units of measurement.

genetics↗

bioRxiv2020

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration

The problem in meat adulteration and food fraud emphasised the requirement of developing accurate analytical approaches for the quantitative detection in helping the control of meat adulteration. In this study, the droplet digital Polymerase Chain Reaction (ddPCR) assays to quantify the ratios of pork DNA to the total amount of meat DNA were developed by challenging against DNA extracted from a range of gravimetrically prepared matrices of pork in beef. A single copy nuclear DNA gene, {beta}-actin, was employed as a target gene, accompanied with myostatin gene as a cross species target for mammal and poultry meat background in order to quantifying approach. All the developed assays, singleplex, duplex and triplex did not show significant difference in quantification of pork content in beef background and demonstrated a good and comparable performance to the mass fractions. The singleplex assay provided more biases than the other two assays when performing with a low concentration of target species. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and running time. All proposed methods allowed us to quantify pork addition in beef with a limit of quantification (LOQ) estimated at 0.1% (w/w) and a limit of detection (LOD) down to 0.01% (w/w). The developed triplex assay was also tested with commercial processed foods and showed the ability to determine not only the presence of particular pork or beef but also the quantitative purpose directly without standard curves. Hence, the developed ddPCR assays demonstrated a good trueness and precision of the methods in quantifying pork or beef content for meat adulteration. It is expected that these developed approaches can be applied to help regulators to confidently enforce food labelling obligations.

molecular biology↗