Last data update: May 20, 2024. (Total: 46824 publications since 2009)
Records 1-3 (of 3 Records) |
Query Trace: Hutchins RJ[original query] |
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Practical Guidance to Implementing Quality Management Systems in Public Health Laboratories Performing Next Generation Sequencing: Personnel, Equipment, and Process Management (Phase 1).
Hutchins RJ , Phan KL , Saboor A , Miller JD , Muehlenbachs A . J Clin Microbiol 2019 57 (8) Quality standards as part of an effective quality management system (QMS) are the cornerstone for generating high-quality test results. Next-generation sequencing (NGS) has the potential to improve both clinical diagnostics and public health surveillance efforts in multiple areas, including infectious diseases. However, the laboratories adopting NGS methods face significant challenges due to the complex and modular process design. This document summarizes the first phase of quality system guidance developed by the Centers for Disease Control and Prevention (CDC) NGS Quality Workgroup. The quality system essentials of personnel, equipment, and process management (quality control and validation) were prioritized based on a risk assessment using information gathered from participating CDC laboratories. Here, we present a prioritized QMS framework, including procedures and documentation tools, to assist laboratory implementation and maintenance of quality practices for NGS workflows. |
Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5'nuclease assay for Ebola virus
Stephens KW , Hutchins RJ , Dauphin LA . Mol Cell Probes 2010 24 (6) 370-5 Selection of optimal reaction master mix reagents is essential to obtain the best performance with diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Every year the number of commercially available master mix kits increases, so it is prudent to periodically evaluate kits on the market. In this study we evaluated five commercial real-time RT-PCR master mix kits, the RealMasterMix RT-PCR ROX kit, the AgPath-ID One-Step RT-PCR kit, the SuperScript III Platinum One-step Quantitative RT-PCR system, the QuantiTect Probe RT-PCR kit, and the LightCycler RNA HybProbe amplification kit, using a 5'nuclease assay for Ebola virus. The kits were evaluated using the manufacturer's recommended conditions, as well as conditions which have been used with the Ebola virus assay during outbreaks. When evaluated for use in Ebola virus RNA detection, the AgPath-ID kit resulted in the greatest sensitivity in comparison to the other four kits. The efficacy of the AgPath-ID kit was instrument-independent in the five real-time PCR platforms tested. This study demonstrated that Ebola virus RNA detection was not equivalent among the master mix reagents studied and, thus, that this variable can affect real-time RT-PCR assay sensitivity. Furthermore, this study rates the master mix reagents for their suitability, providing diagnostic laboratories the option to select from these kits to suit their specific laboratory needs for real-time RT-PCR. |
Evaluation of automated and manual commercial DNA extraction methods for the recovery of Brucella spp. DNA from suspensions and spiked swabs
Dauphin LA , Hutchins RJ , Bost LA , Bowen MD . J Clin Microbiol 2009 47 (12) 3920-6 This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in PBS suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing varying throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA Sample Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit, and the UltraClean Microbial DNA Isolation Kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure and the MagNA Pure Compact kits performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and thus that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella. |
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