Search results for " PCR"
showing 10 items of 153 documents
Comparative multiplex dosage analysis in spinocerebellar ataxia type 2 patients.
2013
We developed a new application of comparative multiplex dosage analysis (CMDA) for evaluation of the ataxin 2 gene. Expansions of the triplet CAG can cause spinocerebellar ataxia type 2 (SCA2), a neurodegenerative disease with an autosomal-dominant mode of inheritance. Molecular diagnosis of SCA2 is routinely based on the use of conventional PCR to detect the CAG expansion. However, PCR does not amplify an allele with an expansion of many triplets (>80), which is typically found in infantile and juvenile forms of SCA2, thus leading to false negatives. We propose the analysis of the ATXN2 gene by CMDA to complement existing methods currently used for the detection of large expansions of the …
All-Food-Seq (AFS): a quantifiable screen for species in biological samples by deep DNA sequencing.
2013
Background DNA-based methods like PCR efficiently identify and quantify the taxon composition of complex biological materials, but are limited to detecting species targeted by the choice of the primer assay. We show here how untargeted deep sequencing of foodstuff total genomic DNA, followed by bioinformatic analysis of sequence reads, facilitates highly accurate identification of species from all kingdoms of life, at the same time enabling quantitative measurement of the main ingredients and detection of unanticipated food components. Results Sequence data simulation and real-case Illumina sequencing of DNA from reference sausages composed of mammalian (pig, cow, horse, sheep) and avian (c…
Seasonal trend of Anisakidae infestation in South Mediterranean bluefish
2020
A total of 1104 fish samples from markets of Sicily were analysed for the detection and species identification of Anisakidae nematodes. The preliminary analysis of the fish samples showed the presence of 2459 larvae. All the fish species revealed different prevalence of infestation, with a maximum of 100% for Lepidopus caudatus and a minimum of 4.5% in Sardina pilchardus. The 80% of the larvae examined by PCR-RFLP analysis belonged to Anisakis pegreffii species. The seasonal infestation trend of Anisakis was evaluated in all the fish sample examined. The results of the seasonal infestation trend showed a marked connection with the ecological aspects of the fish species examined. As far as w…
Comparison of the performance of 2 commercial multiplex PCR platforms for detection of respiratory viruses in upper and lower tract respiratory speci…
2015
The performance of the CLART® PneumoVir system with that of the Luminex xTAG RVP Fast v1 assay for detection of most common respiratory viruses in upper and lower tract respiratory specimens (n = 183) from unique patients with influenza-like syndrome or lower tract respiratory infection. Nested PCR coupled to automated sequencing was used for resolution of discrepancies. Fully concordant results were obtained for a total of 122 specimens, whereas 56 specimens gave partially (n = 21) or fully discordant (n = 35) results (Kappa coefficient, 0.62). The overall specificity of the Luminex xTAG RVP Fast v1 assay was slightly higher than that of the CLART® PneumoVir assay for human bocavirus, infl…
Nation-wide study of the occurrence of Listeria monocytogenes in French soils using culture-based and molecular detection methods
2013
Identifiant HAL : hal-01120618; International audience; Soil is a potential reservoir of human pathogens and a possible source of contamination of animals, crops and water. In order to study the distribution of Listeria monocytogenes in French soils, a real-time PCR TaqMan assay targeting the phosphoribosylpyrophosphate synthetase (prs) gene of L. monocytogenes was developed for the specific detection and quantification of this bacterium within a collection of 1315 soil DNAs originated from the French Soil Quality Monitoring Network. The prs real-time PCR TaqMan assay was specific for L. monocytogenes and could quantify accurately down to 104L. monocytogenes per gram of dry soil. Among the …
Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR
2019
The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematolog…
The use of multiplex PCR to detect and differentiate food- and beverage-associated microorganisms: a review.
2007
Regarding food safety, rapid detection of microbial species is crucial to develop effective preventive and/or adjustment measures. Classical methods for determining the presence of certain species are time-consuming and labor-intensive, hence, molecular methods, which offer speed, sensitivity and specificity, have been developed to address this problem. Multiplex PCR (MPCR) is widely applied in the various fields of microbiology for the rapid differentiation of microbial species without compromising accuracy. This paper describes the method and reports on the state-of-the-art application of this technique to the identification of microorganisms vehiculated with foods and beverages. The iden…
SCAR-based real time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils.
2006
Strains of Trichoderma spp. are known for their antagonistic properties against plant pathogens, some are already on the market, others are under development. In order to launch a strain on the market its perfect identification at the species and strain levels is needed. The aim of this study is to (i) design a SCAR marker for specific identification of strain T1 of Trichoderma atroviride and (ii) monitor population dynamics of this strain in soil by real time PCR. A primer pair targeting a 141-bp fragment enabled specific detection of this strain without cross detection of autochthonous populations of Trichoderma in several field soils. In two soils, population dynamics assessed by real ti…
Freezing and storage at -20 °C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis.
2014
Equipe EA MERS; International audience; Nucleic acid-based testing has become crucial for toxoplasmosis diagnosis. For retrospective (forensic or scientific) studies, optimal methods must be employed for DNA long-term storage. We compared Toxoplasma gondii detection before and after DNA storage using real-time PCR. No significant differences were found depending on duration or storage conditions at -20 °C or -80 °C.
Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory t…
2013
Staphylococcus aureus is a significant cause of hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. We used the fibronectin-binding protein A gene (fnbA) for the species-specific and quantitative detection of S. aureus directly from lower respiratory tract (LRT) specimens by a Taq Man real time PCR. For this reason, a total of 269 lower respiratory tract (LRT) specimens collected from patients with hospital-acquired pneumonia were assayed. Amplification of fnbA in serial dilutions ranged from 10(9) CFU/ ml to 10(2) CFU/ml. Standard curve of triplicate every dilution had slope 3.34±0.1 and R2>0.99 with SD 0.1. Based on these data, the sensitivity and specif…