Search results for: FTA PURIFICATION REAGENT
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FTA Cards Facilitate Storage, Shipment, and Detection of Arboviruses in InfectedCollected in Adult Mosquito Traps.AbstractThe utility of applying infectedto Flinders Technology Associates (FTA) cards for storage, transport, and detection of dengue, Zika, and Barmah Forest viruses was assessed in laboratory-based experiments. The mosquitoes had been removed from GravidTraps maintained under conditions of high temperature and humidity. RNA of all viruses could be detected in infected mosquitoes on FTA cards either individually or in pools with uninfected mosquitoes, and stored for up to 28 days. Importantly, there was only a minimal decrease in RNA levels in mosquitoes between days 0 and 28, indicating that viral RNA was relatively stable on the cards. FTA cards thus provide a mechanism for storing potentially infected mosquitoes collected in the field and transporting them to a central diagnostic facility for virus detection.
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A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classiccards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelexresin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent.
1125 related Products with: A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.AccuPrep Genomic DNA Extr Recombinant Tick-Borne En Recombinant Tick-Borne En Recombinant Tick-Borne En Recombinant Tick-Borne En Recombinant Tick-Borne En Recombinant Tick-Borne En AccuPrep GMO DNA Extracti AccuPrep Genomic DNA Extr Kits for 96 well Plate Va AccuPrep Stool DNA Extrac AccuPrep Stool DNA Extrac
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ASSESSMENT OF THREE BLOOD GENOMIC-DNA PREPARATION METHODS FOR MALARIA MOLECULAR DIAGNOSIS.Species-specific PCR techniques are highly sensitive and reliable alternatives to classical methods for malaria diagnosis and speciation, especially in endemic regions under advanced control or elimination programs where asymptomatic and low-density infections are increasingly reported. Nevertheless, the performance of these techniques is directly affected by the quality of isolated DNA templates. A Plasmodium falciparum/vivax-specific diagnostic Nested-PCR (Pƒ/Pv N-PCR) was used to assess three DNA preparation methods, Qiagen® Mini-Chromatographic kit (QIAmp®) and Jena-Biosciences® DNA isolation kit (JB®) for genomic DNA extraction from EDTA-preserved whole blood samples, and Whatman-FTA® purification reagent (FTA®) for DNA preparation from dry blood spots (DBS) collected onto FTA®- cards. A total of 84 out of 137 blood specimens collected from malaria suspicious febrile patients who visited five health care centres in south-western endemic localities of Saudi Arabia were found P. falciparum positive by at least one method. Among these, only 76 (90%) were reported P. falciparum malaria positive by two expert microscopists. No other species of Plasmodium were detected. Pƒ/Pv N-PCR revealed 84/84 (100%), 75/84 (89%), and 81 (96%) P. falciparum positive samples using DNA templates prepared by QIAmp®, JB®, and FTA® purification methods, respectively. Therefore, Pƒ/Pv N-PCR, when applied to QIAmp® DNA templates showed to be a highly sensitive diagnostic method, particularly useful for submicroscopic specimens from clinically malaria suspicious patients in endemic areas. On the other hand, Pƒ/Pv N-PCR of FTA®-DBS DNA templates revealed 5 positive cases missed by microscopy, encouraging its use as an affordable field semi-adapted protocol for malaria active screening, especially in remqte rural regions with limited laboratory infrastructure.
1917 related Products with: ASSESSMENT OF THREE BLOOD GENOMIC-DNA PREPARATION METHODS FOR MALARIA MOLECULAR DIAGNOSIS.AccuPrep Genomic DNA Extr ProPrep™ Genomic XL-10 Plant DNA Preparation Kit Growth Differentiation Fa DNA (cytosine 5) methyltr Genomic DNA Isolation Kit JETFLEX Genomic DNA Purif JETFLEX Genomic DNA Purif Rat Genomic DNA AccuPrep Genomic DNA Extr Kits for 96 well Plate Va ExiPrep Bacteria Genomic
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RNA isolation from bloodstains collected on FTA cards - application in clinical and forensic genetics.Aim of the study: In recent years, RNA analysis has been increasingly used in clinical and forensic genetics. Nevertheless, a major limitation of RNA-based applications is very low RNA stability in biological material, due to the RNAse activity. This highlights the need for improving the methods of RNA collection and storage. Technological approaches such as FTA Classic Cards (Whatman) could provide a solution for the problem of RNA degradation. However, different methods of RNA isolation from FTA cards could have diverse effects on RNA quantity and quality. The purpose of this research was to analyze the utility of three different methods of RNA isolation from peripheral blood collected on FTA Classic Cards (Whatman). The study also aimed at assessing RNA stability in bloodstains deposited on FTA cards. Material and methods: The study was performed on peripheral bloodstains collected from 59 individuals on FTA Classic Cards (Whatman). RNA was isolated with High Pure RNA Isolation Kit (Roche Diagnostics), Universal RNA/miRNA Purification (EURx) and TRIzol Reagent (Life Technologies). RNA was subjected to quantitative analysis followed by reverse transcription and Real - Time PCR reaction. Results: The study has shown that FTA Classic Cards (Whatman) are useful tools for storing bloodstains at room temperature for RNA analysis. Moreover, the method of RNA extraction employing TRIzol Reagent (Life Technologies) provides the highest efficiency and reproducibility for samples stored for no more than 2 years. Conclusions: The FTA cards are suitable for collecting and storing bloodstains for RNA analysis in clinical and forensic genetics.
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Differences in gluten metabolism among healthy volunteers, coeliac disease patients and first-degree relatives.Coeliac disease (CD) is an immune-mediated enteropathy resulting from exposure to gluten in genetically predisposed individuals. Gluten proteins are partially digested by human proteases generating immunogenic peptides that cause inflammation in patients carrying HLA-DQ2 and DQ8 genes. Although intestinal dysbiosis has been associated with patients with CD, bacterial metabolism of gluten has not been studied in depth thus far. The aim of this study was to analyse the metabolic activity of intestinal bacteria associated with gluten intake in healthy individuals, CD patients and first-degree relatives of CD patients. Faecal samples belonging to twenty-two untreated CD patients, twenty treated CD patients, sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD were tested for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten in faeces. We detected faecal peptidasic activity against the gluten-derived peptide 33-mer. CD patients showed differences in faecal glutenasic activity (FGA), faecal tryptic activity (FTA), SCFA and faecal gluten content with respect to healthy volunteers. Alterations in specific bacterial groups metabolising gluten such as Clostridium or Lactobacillus were reported in CD patients. Relatives showed similar parameters to CD patients (SCFA) and healthy volunteers (FTA and FGA). Our data support the fact that commensal microbial activity is an important factor in the metabolism of gluten proteins and that this activity is altered in CD patients.
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An evaluation of direct PCR amplification.To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol.
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Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.
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Rapid Sanger sequencing of the 16S rRNA gene for identification of some common pathogens.Conventional Sanger sequencing remains time-consuming and laborious. In this study, we developed a rapid improved sequencing protocol of 16S rRNA for pathogens identification by using a new combination of SYBR Green I real-time PCR and Sanger sequencing with FTA® cards. To compare the sequencing quality of this method with conventional Sanger sequencing, 12 strains, including three kinds of strains (1 reference strain and 3 clinical strains, which were previously identified by biochemical tests), which have 4 Pseudomonas aeruginosa, 4 Staphyloccocus aureus and 4 Escherichia coli, were targeted. Additionally, to validate the sequencing results and bacteria identification, expanded specimens with 90 clinical strains, also comprised of the three kinds of strains which included 30 samples respectively, were performed as just described. The results showed that although statistical differences (P<0.05) were found in sequencing quality between the two methods, their identification results were all correct and consistent. The workload, the time consumption and the cost per batch were respectively light versus heavy, 8 h versus 11 h and $420 versus $400. In the 90 clinical strains, all of the Pseudomonas aeruginosa and Staphyloccocus aureus strains were correctly identified, but only 26.7% of the Escherichia coli strains were recognized as Escherichia coli, while 33.3% as Shigella sonnei and 40% as Shigella dysenteriae. The protocol described here is a rapid, reliable, stable and convenient method for 16S rRNA sequencing, and can be used for Pseudomonas aeruginosa and Staphyloccocus aureus identification, yet it is not completely suitable for discriminating Escherichia coli and Shigella strains.
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Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field.A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 μg/mL for A. invadans compared to 56 μg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10(-11) dilution compared to a 10(-8) dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8°C. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.
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Statistical adjustment of genotyping error in a case-control study of childhood leukaemia.Genotyping has become more cost-effective and less invasive with the use of buccal cell sampling. However, low or fragmented DNA yields from buccal cells collected using FTA cards often requires additional whole genome amplification to produce sufficient DNA for genotyping. In our case-control study of childhood leukaemia, discordance was found between genotypes derived from blood and whole genome amplified FTA buccal DNA samples. We aimed to develop a user-friendly method to correct for this genotype misclassification, as existing methods were not suitable for use in our study.
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