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We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching process for PCR with a fluorescence analog of Beer’s Law, the approach reduces bias and quantifies uncertainty in fluorescence. As the two-type branching process distinguishes between complementary strands of DNA, it allows for a stoichiometric description of reactions between fluorescent probes and DNA and can capture the initial conditions encountered in assays targeting RNA. Analysis of the expected copy-number identifies additional dynamics that occur at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and strand-specific amplification (i.e., if one strand is synthesized more efficiently than its complement). Linking the branching process to fluorescence by the Beer’s Law analog allows for an a priori description of background fluorescence. It also enables uncertainty quantification (UQ) in fluorescence which, in turn, leads to analytical relationships between amplification efficiency (probability) and limit of detection. This work sets the stage for UQ-PCR, where both the input copy-number and its uncertainty are quantified from fluorescence kinetics. [ABSTRACT FROM AUTHOR]

Motivated by the current COVID-19 health crisis, we consider data analysis for quantitative polymerase chain-reaction (qPCR) measurements. We derive a theoretical result specifying the conditions under which all qPCR amplification curves (including their plateau phases) are identical up to an affine transformation, i.e. a multiplicative factor and horizontal shift. We use this result to develop a data analysis procedure for determining when an amplification curve exhibits characteristics of a true signal. The main idea behind this approach is to invoke a criterion based on constrained optimization that assesses when a measurement signal can be mapped to a master reference curve. We demonstrate that this approach: (i) can decrease the fluorescence detection threshold by up to a decade; and (ii) simultaneously improve confidence in interpretations of late-cycle amplification curves. Moreover, we demonstrate that the master curve is transferable reference data that can harmonize analyses between different labs and across several years. Application to reverse-transcriptase qPCR measurements of a SARS-CoV-2 RNA construct points to the usefulness of this approach for improving confidence and reducing limits of detection in diagnostic testing of emerging diseases. [ABSTRACT FROM AUTHOR]

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA.The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for “haploid human genome equivalent”; there are two HHGE per diploid human genome. [ABSTRACT FROM AUTHOR]

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles.PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA [ABSTRACT FROM AUTHOR]

Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 μmol/L (47.5 μmol/L to 51.5 μmol/L) and 77.0 μmol/L (54.3 μmol/L to 99.4 μmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself. [ABSTRACT FROM AUTHOR]