Development and Analytical Validation of a DNA Dual-Strand Approach for the US Food and Drug Administration-Approved Next-Generation Sequencing-Based Praxis Extended RAS Panel for Metastatic Colorectal Cancer Samples.

A next-generation sequencing method was developed that can distinguish single-stranded modifications from low-frequency somatic mutations present on both strands of DNA in formalin-fixed paraffin-embedded colorectal cancer samples. We applied this method for analytical validation of the Praxis Extended RAS Panel, a US Food and Drug Administration-approved companion diagnostic for panitumumab, on the Illumina MiSeqDx platform. With the use of the TruSeq amplicon workflow, both strands of DNA from the starting material were interrogated independently. Mutations were reported only if found on both strands; artifacts usually present on only one strand would not be reported. A total of 56 mutations were targeted within the KRAS and NRAS genes. A minimum read depth of 1800× per amplicon is required per sample but averaged >30,000× at maximum multiplexing levels. Analytical validation studies were performed to determine the simultaneous detection of mutations on both strands, reproducibility, assay detection level, precision of the assay across various factors, and the impact of interfering substances. In conclusion, this assay can clearly distinguish single-stranded artifacts from low-frequency mutations. Furthermore, the assay is accurate, precise, and reproducible, can achieve consistent detection of a mutation at 5% mutation frequency, exhibits minimal impact from tested interfering substances, and can simultaneously detect 56 mutations in a single run using 10 samples plus controls.

Effects of Novel 3-dimensional Antibiotic-containing Electrospun Scaffolds on Dentin Discoloration.

INTRODUCTION: Although intracanal application of the triple antibiotic paste (TAP) may offer advantages (eg, disinfection), this practice has been associated with significant drawbacks, including tooth discoloration. In this study, the color change of dentin was monitored during treatment with distinct TAP pastes and novel tubular-shaped 3-dimensional electrospun scaffolds containing minocycline (MINO) or doxycycline (DOX). METHODS: Two TAP pastes (TAPMINO [MINO, metronidazole, and ciprofloxacin] and TAPDOX [DOX, metronidazole, and ciprofloxacin]), 4 scaffold-based groups containing MINO or DOX at distinct concentrations, 1 antibiotic-free scaffold, and 1 untreated group (control) were investigated. Human canines were sectioned at the cementoenamel junction and tubular-shaped scaffolds or paste were placed into the root canals and sealed. Color measurements (CIEL(*)a(*)b(*) parameters) were performed at baseline and after 1, 3, 7, 14, 21, and 28 days. Color changes were expressed as ΔE(*) values. In addition, scanning electron microscopy and energy-dispersive X-ray spectroscopy were also performed on the specimens after treatment. Data were analyzed using repeated measures analysis of variance (alpha = 0.05). RESULTS: All antibiotic-containing groups led to greater discoloration than the antibiotic-free groups. A severe discoloration occurred after 1 day. At the end of the experiment, antibiotic-treated samples exhibited crusts/agglomerates over the dentin surface, which totally or partially obliterated the dentinal tubules. The presence of MINO resulted in a greater color change than DOX. CONCLUSIONS: Scaffolds containing MINO or DOX produced similar color change to dentin when compared with their respective TAP systems, although DOX-related discoloration was less pronounced.