Search results for "DNA extraction"
showing 10 items of 66 documents
Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid.
2018
International audience; Objectives - Molecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays.Methods - In this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS and Freedom EVO VacS, Tecan), compared with three other …
Multiplex Detection of Aspergillus Species
2016
Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied …
Molecular detection of Borrelia burgdorferi sensu lato – An analytical comparison of real-time PCR protocols from five different Scandinavian laborat…
2017
Introduction Lyme borreliosis (LB) is the most common tick transmitted disease in Europe. The diagnosis of LB today is based on the patient A s medical history, clinical presentation and laboratory findings. The laboratory diagnostics are mainly based on antibody detection, but in certain conditions molecular detection by polymerase chain reaction (PCR) may serve as a complement. Aim The purpose of this study was to evaluate the analytical sensitivity, analytical specificity and concordance of eight different real-time PCR methods at five laboratories in Sweden, Norway and Denmark. Method Each participating laboratory was asked to analyse three different sets of samples (reference panels; a…
Validation and implementation of a commercial real-time PCR assay for direct detection of Candida auris from surveillance samples.
2021
Background Rapid and reliable laboratory methods are required for detecting the nosocomial yeast Candida auris. AurisID® (Olm Diagnostics, England) is a real-time PCR assay approved for detecting C. auris in fungal cultures as well as directly from blood samples, involving a nucleic acid extraction as a prior step. Objectives The purpose of this study is to validate the AurisID® kit for direct detection of C. auris from surveillance samples without prior DNA extraction and to analyze the results of implementing this methodology to our daily laboratory routine protocol for C. auris surveillance studies. Methods Our PCR method using the AurisID® kit was compared with our routine protocol, con…
Impact of temperature and soil type on Mycobacterium bovis survival in the environment
2017
Mycobacterium bovis, the causative agent of the bovine tuberculosis (bTB), mainly affects cattle, its natural reservoir, but also a wide range of domestic and wild mammals. Besides direct transmission via contaminated aerosols, indirect transmission of the M. bovis between wildlife and livestock might occur by inhalation or ingestion of environmental substrates contaminated through infected animal shedding. We monitored the survival of M. bovis in two soil samples chosen for their contrasted physical and-chemical properties (i.e. pH, clay content). The population of M. bovis spiked in sterile soils was enumerated by a culture-based method after 14, 30, 60, 90, 120 and 150 days of incubation…
Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor
2017
Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was an…
Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quanti…
2011
ABSTRACT Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) …
Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues
1990
DNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at -25 degrees C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day postmortem. However, more than 90% of DNA samples up to at least 3 days postmortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed goo…
Evaluation of a new pooling strategy based on leukocyte count for rapid quantification of allele frequencies.
2007
Abstract Background: Allele frequencies of single-nucleotide polymorphisms (SNPs) can be quantified from DNA pools. The conventional preparation of DNA pools requires DNA isolation and quantification for each blood sample. We hypothesized that pooling of whole blood samples according to their leukocyte count, which determines DNA content, would be as reliable as the conventional pooling method but much less tedious to perform. Methods: We collected 100 whole blood samples and measured the leukocyte count. Samples were frozen until further use. After thawing, pools were generated by combining aliquots containing an equal number of leukocytes. In parallel, DNA was extracted from another aliqu…
An Optimized and Rapid DNA Extraction Method From Leaves of Grapevine Suitable for PCR-DGGE Based Analysis
2010
Molecular techniques and genetic studies require a fair amount of DNA of high quality in order to produce reliable and clear results (Li et al., 2007). In grapevine, the yield and quality of DNA can be significantly affected by secondary metabolites such as polyphenols, polysaccharides and tannins, that may be consistently abundant during the different stages of leaf development (Iandolino et al., 2004). The development of a simple, rapid and reliable method for the extraction of genomic DNA from grape leaves collected at several stages during development, as well as from healthy grape leaves and leaves infected by pathogenic microorganisms, was the main objective of this study. The protoco…