Evaluation of a new automated, standardized generic nucleic acid extraction method (total nucleic acid isolation kit) used in combination with cytomegalovirus DNA quantification by COBAS AMPLICOR CMV MONITOR.
Ontology highlight
ABSTRACT:
SUBMITTER: Gartner BC
PROVIDER: S-EPMC497653 | biostudies-other | 2004 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Automated nucleic acid extraction from primary (raw) sputum continues to be a significant technical challenge for molecular diagnostics. In this work, we developed a prototype open-architecture, automated nucleic acid workstation that includes a mechanical homogenization and lysis function integrated with heating and TruTip purification; optimized an extraction protocol for raw sputum; and evaluated system performance on primary clinical specimens. Eight samples could be processed within 70 min. The system efficiently homogenized primary sputa and doubled nucleic acid recovery relative to an automated protocol that did not incorporate sample homogenization. Nucleic acid recovery was at least five times higher from raw sputum as compared to that of matched sediments regardless of smear or culture grade, and the automated workstation reproducibly recovered PCR-detectable DNA to at least 80 CFU mL-1 raw sputum. M. tuberculosis DNA was recovered and detected from 122/123 (99.2%) and 124/124 (100%) primary sputum and sediment extracts, respectively. There was no detectable cross-contamination across 53 automated system runs and amplification or fluorescent inhibitors (if present) were not detectable. The open fluidic architecture of the prototype automated workstation yields purified sputum DNA that can be used for any molecular diagnostic test. The ability to transfer TruTip protocols between personalized, on-demand pipetting tools and the fully automated workstation also affords public health agencies an opportunity to standardize sputum nucleic acid sample preparation procedures, reagents, and quality control across multiple levels of the health care system.
| S-EPMC6033430 | biostudies-literature
Project description:Quantitation of cytomegalovirus (CMV) viral load in the transplant patients has become a standard practice for monitoring the response to antiviral therapy. The cut-off values of CMV viral load assays for preemptive therapy are different due to the various assay designs employed. To establish a sensitive and reliable diagnostic assay for preemptive therapy of CMV infection, two commercial automated platforms including m2000sp extraction system integrated the Abbott RealTime (m2000rt) and the Roche COBAS AmpliPrep for extraction integrated COBAS Taqman (CAP/CTM) were evaluated using WHO international CMV standards and 110 plasma specimens from transplant patients. The performance characteristics, correlation, and workflow of the two platforms were investigated. The Abbott RealTime assay correlated well with the Roche CAP/CTM assay (R2 = 0.9379, P<0.01). The Abbott RealTime assay exhibited higher sensitivity for the detection of CMV viral load, and viral load values measured with Abbott RealTime assay were on average 0.76 log10 IU/mL higher than those measured with the Roche CAP/CTM assay (P<0.0001). Workflow analysis on a small batch size at one time, using the Roche CAP/CTM platform had a shorter hands-on time than the Abbott RealTime platform. In conclusion, these two assays can provide reliable data for different purpose in a clinical virology laboratory setting.
| S-EPMC4975419 | biostudies-literature
Project description:Upstream sample preparation remains the bottleneck for point-of-need nucleic acid testing due to its complexity and time-consuming nature. Sample preparation involves extracting, purifying, and concentrating nucleic acids from various matrices. These processes are critical for ensuring the accuracy and sensitivity of downstream nucleic acid amplification and detection. However, current sample preparation methods are often laboratory-based, requiring specialized equipment, trained personnel, and several hours of processing time. As a result, sample preparation often limits the speed, portability, and cost-effectiveness of point-of-need nucleic acid testing. A universal, field-deployable sample preparation device is highly desirable for this critical need and unmet challenge. Here we reported a handheld, battery-powered, reconfigurable, and field-deployable nucleic acid sample preparation device. A programmable electromagnetic actuator was developed to drive a magnetic robot (ProMagBot) in X/Y 2D space, such that various magnetic bead-based sample preparations can be readily translated from the laboratory to point-of-need settings. The control of the electromagnetic actuator requires only a 3-phase unipolar voltage in X and Y directions, and therefore, the motion space is highly scalable. We validated the ProMagBot device with a model application by extracting HIV viral RNAs from plasma samples using two widely used magnetic bead kits: ChargeSwitch and MagMAX beads. In both cases, the ProMagBot could successfully extract viral RNAs from 50 μL plasma samples containing as low as 102 copies of viral RNAs in 20 minutes. Our results demonstrated the ability of ProMagBot to prepare samples from complex mediums at the point of need. We believe such a device would enable rapid and robust sample preparation in various settings, including resource-limited or remote environments, and accelerate the development of next-generation point-of-need nucleic acid testing.
| S-EPMC11218199 | biostudies-literature
Project description:Microbiome nucleic acid extraction kit model is a Named Entity Recognition (NER) model that identifies and annotates the name of the kits used in extracting microbiome nucleic acids in texts. This is the final model version used to annotate metagenomics publications in Europe PMC and enrich metagenomics studies in MGnify with kits metadata from literature. For more information, please refer to the following blogs: http://blog.europepmc.org/2020/11/europe-pmc-publications-metagenomics-annotations.html https://www.ebi.ac.uk/about/news/service-news/enriched-metadata-fields-mgnify-based-text-mining-associated-publications
2022-02-21 | MODEL2202170007 | BioModels
Project description:Yield and quality are fundamental features for any researchers during nucleic acid extraction. Here, we describe a simplified, semi-unified, effective, and toxic material free protocol for extracting DNA and RNA from different prokaryotic and eukaryotic sources exploiting the physical and chemical properties of nucleic acids. Furthermore, this protocol showed that DNA and RNA are under triple protection (i.e. EDTA, SDS and NaCl) during lysis step, and this environment is improper for RNase to have DNA liberated of RNA and even for DNase to degrade the DNA. Therefore, the complete removal of RNA under RNase influence is achieved when RNase is added after DNA extraction, which gives optimal quality with any protocols. Similarly, DNA contamination in an isolated RNA is degraded by DNase to obtain high-quality RNA. Our protocol is the protocol of choice in terms of simplicity, recovery time, environmental safety, amount, purity, PCR and RT-PCR applicability.
| S-EPMC5361071 | biostudies-literature
Project description:Blood plasma, one of the most studied liquid biopsies, contains various molecules that have biomarker potential for cancer detection, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA). As the vast majority of cell-free nucleic acids in circulation are non-cancerous, a laboratory workflow with a high detection sensitivity of tumor-derived nucleic acids is a prerequisite for precision oncology. One way to meet this requirement is by the combined analysis of cfDNA and cfRNA from the same liquid biopsy sample. So far, no study has systematically compared the performance of cfDNA and cfRNA co-purification to increase sensitivity. First, we set up a framework using digital PCR (dPCR) technology to quantify cfDNA and cfRNA from human blood plasma in order to compare cfDNA/cfRNA co-purification kit performance. To that end, we optimized two dPCR duplex assays, designed to quantify both cfDNA and cfRNA with the same assays, by ensuring that primers and probes are located within a highly abundant exon. Next, we applied our optimized workflow to evaluate the co-purification performance of two manual and two semi-automated methods over a range of plasma input volumes (0.06-4 mL). Some kits result in higher nucleic acid concentrations in the eluate, while consuming only half of the plasma volume. The combined nucleic acid quantification systematically results in higher nucleic acid concentrations as compared to a parallel quantification of cfDNA and cfRNA in the eluate. We provide a framework to evaluate the performance of cfDNA/cfRNA co-purification kits and have tested two manual and two semi-automated co-purification kits in function of the available plasma input amount and the intended use of the nucleic acid eluate. We demonstrate that the combined quantification of cfDNA and cfRNA has a benefit compared to separate quantification. We foresee that the results of this study are instrumental for clinical applications to help increase mutation detection sensitivity, allowing improved disease detection and monitoring.
| S-EPMC9805675 | biostudies-literature
Project description:Cytomegalovirus (CMV) DNA quantitation in clinical specimens is progressively becoming a cornerstone in the diagnosis and management of CMV infection in the immunocompromised host. We evaluated two automated and reproducible PCR tests, the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and the COBAS AMPLICOR CMV Monitor (Roche Diagnostics, Pleasanton, Calif.), for the detection of CMV DNA in blood samples from transplant recipients with CMV infection as determined by shell vial culture. Following a log transformation analysis, the mean CMV DNA in plasma (PL), whole blood (WB), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) using the LightCycler was 6.79 copies per ml, 7.23 copies per ml, 6.38 copies per 2 x 10(6) cells, and 6.27 copies per 2 x 10(6) cells, respectively. This compares to 7.86 copies per ml, 8.37 copies per ml, 7.59 copies per 2 x 10(6) cells, and 7.44 copies per 2 x 10(6) cells, respectively, using COBAS AMPLICOR CMV Monitor. While higher CMV DNA levels were observed for the various blood compartments analyzed using COBAS AMPLICOR CMV Monitor, a high degree of correlation was evident between the two automated systems (jackknife correlation r = PL 0.77 [95% confidence interval (CI); 0.64, 0.90], WB 0.77 [95% CI; 0.62, 0.92], PBL 0.77 [95% CI; 0.67, 0.88], and PBMC 0.81 [95% CI; 0.72, 0.89], all P < 0.001). Therefore, we conclude that either automated diagnostic system is accurate for CMV DNA quantitation.
| S-EPMC88568 | biostudies-literature
Project description:A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.
| S-EPMC120553 | biostudies-literature
Project description:NASA has made great strides in the past five years to develop a suite of instruments for the International Space Station in order to perform molecular biology in space. However, a key piece of equipment that has been lacking is an instrument that can extract nucleic acids from an array of complex human and environmental samples. The Omics in Space team has developed the μTitan (simulated micro(μ) gravity tested instrument for automated nucleic acid) system capable of automated, streamlined, nucleic acid extraction that is adapted for use under microgravity. The μTitan system was validated using a whole cell microbial reference (WCMR) standard comprised of a suspension of nine bacterial strains, titrated to concentrations that would challenge the performance of the instrument, as well as to determine the detection limits for isolating DNA. Quantitative assessment of system performance was measured by comparing instrument input challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next Generation Sequencing. Overall, results indicate that the μTitan system performs equal to or greater than a similar commercially available, earth-based, automated nucleic acid extraction device. The μTitan system was also tested in Yellowstone National Park (YNP) with the WCMR, to mimic a remote setting, with limited resources. The performance of the device at YNP was comparable to that in a laboratory setting. Such a portable, field-deployable, nucleic extraction system will be valuable for environmental microbiology, as well as in health care diagnostics.
| S-EPMC7472602 | biostudies-literature
Project description:The crystallographic structure solution of nucleotides and nucleotide complexes is now commonplace. The resulting electron-density maps are often poorer than for proteins, and as a result interpretation in terms of an atomic model can require significant effort, particularly in the case of large structures. While model building can be performed automatically, as with proteins, the process is time-consuming, taking minutes to days depending on the software and the size of the structure. A method is presented for the automatic building of nucleotide chains into electron density which is fast enough to be used in interactive model-building software, with extended chain fragments built around the current view position in a fraction of a second. The speed of the method arises from the determination of the 'fingerprint' of the sugar and phosphate groups in terms of conserved high-density and low-density features, coupled with a highly efficient scoring algorithm. Use cases include the rapid evaluation of an initial electron-density map, addition of nucleotide fragments to prebuilt protein structures, and in favourable cases the completion of the structure while automated model-building software is still running. The method has been incorporated into the Coot software package.
| S-EPMC4224457 | biostudies-literature