Performance of Spectrophotometric and Fluorometric DNA Quantification Methods

Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric (i.e., a NanoDrop instrument) and three fluorometric (i.e., the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) methods. An analysis of variance (ANOVA) scheme was used to determine the influence of the analyst, the method, and the combination of analyst and method, on DNA quantification. For most samples, the measured DNA concentration was close to or slightly above the concentration of 10 ng/μL as specified by the supplier. Results obtained by the three analysts were equal. However, it was found that, compared to the fluorometric kits, the used spectrophotometric instrument in the case of fish DNA samples tends to overestimate the DNA concentration. Therefore, if sufficient sample volume is available, a combination of a spectrophotometric and a fluorometric method is recommended for obtaining data on the purity and the dsDNA concentration of a sample

A Systematic Review on Commercially Available IntegratedSystems for Forensic DNA Analysis

This systematic review describes and discusses three commercially available integrated systems for forensic DNA analysis, i.e., ParaDNA, RapidHIT, and ANDE. A variety of aspects, such as performance, time-to-result, ease-of-use, portability, and costs (per analysis run) of these three (modified) rapid DNA analysis systems, are considered. Despite their advantages and developmental progress, major steps still have to be made before rapid systems can be broadly applied at crime scenes for full DNA profiling. Aspects in particular that need (further) improvement are portability, performance, the possibility to analyze a (wider) variety of (complex) forensic samples, and (cartridge) costs. Moreover, steps forward regarding ease-of-use and time-to-result will benefit the broader use of commercial rapid DNA systems. In fact, it would be a profit if rapid DNA systems could be used for full DNA profile generation as well as indicative analyses that can give direction to forensic investigators which will speed up investigations

FDA authorized molecular point-of-care SARS-CoV-2 tests: A critical review on principles, systems and clinical performances

Since the start of the COVID-19 pandemic, 10 manufacturers of molecular tests for SARS-CoV-2 have received Emergency Use Authorizations from the U.S. Food and Drug Administration for point-of-care or over the counter use. In this review, the working principle of these tests is described as well as the relevant characteristics (e.g. time-to-result and specimen type). The analytical (e.g. analytical sensitivity) and clinical performance (positive and negative percent agreement) and useability characteristics (e.g. cost, reusability and throughput) of these tests are compared and critically reviewed. Also details for relevant respiratory multiplex assays of these 10 manufacturers are discussed. Critical review of scientific literature on these authorized tests revealed that for many of these tests publications are almost or completely absent, with the exception of two systems. The Xpert Xpress has been thoroughly investigated and good performance has been reported, whereas ID NOW is also well-represented in literature, but has relatively low sensitivity

Performance of Spectrophotometric and Fluorometric DNA Quantification Methods

Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric (i.e., a NanoDrop instrument) and three fluorometric (i.e., the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) methods. An analysis of variance (ANOVA) scheme was used to determine the influence of the analyst, the method, and the combination of analyst and method, on DNA quantification. For most samples, the measured DNA concentration was close to or slightly above the concentration of 10 ng/μL as specified by the supplier. Results obtained by the three analysts were equal. However, it was found that, compared to the fluorometric kits, the used spectrophotometric instrument in the case of fish DNA samples tends to overestimate the DNA concentration. Therefore, if sufficient sample volume is available, a combination of a spectrophotometric and a fluorometric method is recommended for obtaining data on the purity and the dsDNA concentration of a sampl

Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk

The C-type lectin dectin-1 activates the transcription factor NF-kappaB through a Syk kinase-dependent signaling pathway to induce antifungal immunity. Here we show that dectin-1 expressed on human dendritic cells activates not only the Syk-dependent canonical NF-kappaB subunits p65 and c-Rel, but also the noncanonical NF-kappaB subunit RelB. Dectin-1, when stimulated by the beta-glucan curdlan or by Candida albicans, induced a second signaling pathway mediated by the serine-threonine kinase Raf-1, which integrated with the Syk pathway at the point of NF-kappaB activation. Raf-1 antagonized Syk-induced RelB activation by promoting sequestration of RelB into inactive p65-RelB dimers, thereby altering T helper cell differentiation. Thus, dectin-1 activates two independent signaling pathways, one through Syk and one through Raf-1, to induce immune response