MOL-PCR Dotaz Zobrazit nápovědu
Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.
- Klíčová slova
- MOL-PCR, bioterrorism, biothreat bacteria, detection panel, magnetic bead,
- Publikační typ
- časopisecké články MeSH
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.
- MeSH
- infekce yersiniemi diagnóza mikrobiologie MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci přenášené potravou diagnóza mikrobiologie parazitologie MeSH
- Toxoplasma genetika izolace a purifikace MeSH
- toxoplazmóza diagnóza parazitologie MeSH
- Yersinia enterocolitica genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viruses are common causes of food- and waterborne diseases worldwide. Conventional identification of these agents is based on cultivation, antigen detection, electron microscopy, or real-time PCR. Because recent technological advancements in detection methods are focused on fast and robust analysis, a rapid multiplexing technology, which can detect a broad spectrum of pathogenic viruses connected to food or water contamination, was utilized. A new semiquantitative magnetic bead-based multiplex system has been designed for simultaneous detection of several targets in one reaction. The system includes adenoviruses 40/41 (AdV), rotavirus A (RVA), norovirus (NoV), hepatitis E virus (HEV), hepatitis A virus (HAV), and a target for external control of the system. To evaluate the detection system, interlaboratory ring tests were performed in four independent laboratories. Analytical specificity of the tool was tested on a cohort of pathogenic agents and biological samples with quantitative PCR as a reference method. Limit of detection (analytical sensitivity) of 5 × 100 (AdV, HEV, and RVA) and 5 × 101 (HAV and NoV) genome equivalents per reaction was reached. This robust, senstivie, and rapid multiplexing technology may be used to routinely monitor and manage viruses in food and water to prevent food and waterborne diseases.
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
- Publikační typ
- tisková chyba MeSH
AIMS: Only afew gene variants were associated with the response to dipeptidylpeptidase-4 inhibitors (DPP4I). KCNQ1 gene variants were previously related both to type 2 diabetes (T2D) and incretin effect. We hypothesized that T2D related KCNQ1 variants would be associated with smaller glucose-lowering effect of DDP4I. METHODS: We performed a retrospective study in 137 Caucasian subjects with T2D who were followed for 6months after initiation of DPP4I treatment. Genotyping for KCNQ1 rs163184 and rs151290 was performed using PCR-HRMA and PCR-RFLP methods, respectively. The main clinical outcome was reduction in HbA1c (ΔHbA1c) after 6-month DPP4I treatment. RESULTS: KCNQ1 rs163184 T>G variant was associated with the response to DPP4I treatment in genetic additive model (β=-0.30, p=0.022). For each G allele in the rs163184 genotype, we observed a 0.3% (3.3mmol/mol) less reduction in HbA1c during treatment with a DPP4I. Both the GG homozygotes and G-allele carriers had significantly smaller HbA1c reduction in comparison with the TT homozygotes. CONCLUSIONS: KCNQ1 rs163184 T>G variant was associated with a reduced glycaemic response to DPP4I. The difference of 0.6% (6.5mmol/mol) in HbA1c reduction between the TT and GG homozygotes might be of clinical significance if replicated in further studies.
- Klíčová slova
- DPP-4 inhibitors, KCNQ1, Pharmacogenetics, Type 2 diabetes,
- MeSH
- alely MeSH
- diabetes mellitus 2. typu farmakoterapie genetika MeSH
- draslíkový kanál KCNQ1 genetika MeSH
- genotyp MeSH
- glykovaný hemoglobin analýza MeSH
- inhibitory dipeptidylpeptidasy 4 terapeutické užití MeSH
- krevní glukóza analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- polymorfismus genetický genetika MeSH
- retrospektivní studie MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- draslíkový kanál KCNQ1 MeSH
- glykovaný hemoglobin MeSH
- inhibitory dipeptidylpeptidasy 4 MeSH
- krevní glukóza MeSH
We revealed four statistically significant correlations related to inter-simple-sequence repeat (ISSR) patterns: (1) between thermodynamic free energy DeltaG degrees of ISSR primer sequence and PCR reamplification intensity (dA(i)), (2) between free energy DeltaG degrees of ISSR primer sequence and PIC coefficient quantifying the polymorphism of ISSR patterns, (3) and (4) between free energy DeltaG degrees of anchor sequence of primer and the number of total, and polymorphic bands in ISSR patterns, respectively. Methodological recommendations for effective ISSR primer design were inferred based on revealed correlations. In particular, free energy of ISSR primer sequence is recommended to be DeltaG degrees > 160 kJ/mol of interaction and free energy of flanking anchor sequence in primer to be around DeltaG degrees = 28 kJ/mol of interaction to produce ISSR patterns displaying maximum polymorphism of flax germplasm.
- MeSH
- DNA primery MeSH
- DNA rostlinná chemie MeSH
- len klasifikace genetika MeSH
- polymerázová řetězová reakce metody MeSH
- repetitivní sekvence nukleových kyselin * MeSH
- stabilita léku MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
- DNA rostlinná MeSH
Gliptins act by increasing endogenous incretin levels. Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic peptide receptor (GIPR) are their indirect drug targets. Variants of GLP1R and GIPR have previously been associated with the incretin effect. The aim of the present pilot study was to examine associations of the GLP1R and GIPR gene variants with the glycaemic response to gliptins. A total of 140 consecutive patients with type 2 diabetes were followed-up 6 months after initiation of gliptin treatment. GLP1R rs6923761 (Gly168Ser) and GIPR rs10423928 genotyping was performed using real-time PCR, with subsequent high-resolution melting analysis. The main study outcome was reduction in glycated haemoglobin (HbA1c) after treatment. GLP1R Gly168Ser variant was significantly associated with reduction in HbA1c in an additive model (β = -0.33, p = 0.011). The mean reduction in HbA1c in Ser/Ser homozygotes was significantly lower compared with Gly-allele carriers [0.12 ± 0.23% vs. 0.80 ± 0.09% (1.3 ± 2.5 mmol/mol vs. 8.7 ± 1.0 mmol/mol); p = 0.008]. In conclusion, GLP1R missense variant was associated with a reduced response to gliptin treatment. The genotype-related effect size of ∼0.7% (8 mmol/mol) is equal to an average effect of gliptin treatment and makes this variant a candidate for use in precision medicine.
- Klíčová slova
- GLP1R polymorphism, dipeptidyl peptidase inhibitor, oral antidiabetic drug, pharmacogenetics,
- MeSH
- diabetes mellitus 2. typu farmakoterapie genetika metabolismus MeSH
- farmakogenomické varianty MeSH
- genotyp MeSH
- glykovaný hemoglobin metabolismus MeSH
- individualizovaná medicína MeSH
- inhibitory dipeptidylpeptidasy 4 terapeutické užití MeSH
- krevní glukóza metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- missense mutace MeSH
- pilotní projekty MeSH
- receptor pro glukagonu podobný peptid 1 genetika MeSH
- receptory gastrointestinálních hormonů genetika MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- gastric inhibitory polypeptide receptor MeSH Prohlížeč
- GLP1R protein, human MeSH Prohlížeč
- glykovaný hemoglobin MeSH
- hemoglobin A1c protein, human MeSH Prohlížeč
- inhibitory dipeptidylpeptidasy 4 MeSH
- krevní glukóza MeSH
- receptor pro glukagonu podobný peptid 1 MeSH
- receptory gastrointestinálních hormonů MeSH
Physical adsorption of methane in purely siliceous molecular sieves prepared by a recently discovered synthetic pathway using 2D zeolites as nanoscale building blocks has been investigated by means of combined experimental and theoretical approaches. The DFT/CC-based method has been tested on ADOR zeolites of the UTL family and a few experimentally well-characterized siliceous zeolites. Excellent agreement between theoretical and experimental heats of adsorption has been found for OKO, PCR, MFI, CHA and AEI zeolites. The observed discrepancy for the UTL germanosilicate (2 kJ mol-1) has been plausibly explained using a simple model of D4R defects. The proposed methodology can be used as a reliable characterization tool for newly synthesized silica nanomaterials.
- Publikační typ
- časopisecké články MeSH
A Gram-variable, facultatively anaerobic, endospore-forming bacterium was isolated from surface-sterilized seeds of the garden pea and characterized with phenotypic and molecular methods. A PCR with the Paenibacillus-specific primer PAEN515F and the 16S rRNA gene sequence indicated that strain C/2T belongs to the genus Paenibacillus and is closely related to Paenibacillus phyllosphaerae (94.0 % sequence similarity). Strain C/2T generated a unique phenotypic profile, in particular for the production of acid from substrates. The DNA G+C content (50.8 mol%) and the major fatty acid (anteiso-C(15 : 0)) are consistent with the genus Paenibacillus. DNA-DNA hybridization distinguished strain C/2T from other phylogenetically related Paenibacillus species and, therefore, strain C/2T (=CCM 4839T=LMG 23002T) is here described as the type strain of a novel species, for which the name Paenibacillus mendelii sp. nov. is proposed.
- MeSH
- DNA bakterií analýza chemie izolace a purifikace MeSH
- fylogeneze MeSH
- geny rRNA MeSH
- grampozitivní bakterie klasifikace izolace a purifikace fyziologie MeSH
- hrách setý mikrobiologie MeSH
- hybridizace nukleových kyselin MeSH
- mastné kyseliny analýza izolace a purifikace MeSH
- molekulární sekvence - údaje MeSH
- ribozomální DNA chemie izolace a purifikace MeSH
- RNA ribozomální 16S analýza genetika MeSH
- sekvenční analýza DNA MeSH
- semena rostlinná mikrobiologie MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- mastné kyseliny MeSH
- ribozomální DNA MeSH
- RNA ribozomální 16S MeSH
Metal-related genes (afe_0654, afe_0671, afe_0674, afe_1143, afe_1144, and afe_2126) were cloned to identify whether those genes existed in Acidithiobacillus ferrooxidans strain DC (A. ferrooxidans DC). The deduced amino acid sequences of those genes were analyzed by bioinformatics. The tolerance levels of A. ferrooxidans DC to Mn(2+), Zn(2+), and Cd(2+) were determined, which were 0.52, 0.42, and 0.16 mol/L for ferrous iron-grown cells and 0.38, 0.18, and 0.08 mol/L for sulfur-grown cells, respectively. Real-time quantitative PCR was employed to analyze the transcriptional levels of the metal-related genes when ferrous iron- and sulfur-grown cells of A. ferrooxidans DC, respectively, exposed to Mn(2+), Zn(2+), and Cd(2+). The metal-related genes were up-regulated when A. ferrooxidans DC exposed to Mn(2+). When A. ferrooxidans DC exposed to Zn(2+), the metal-related genes were up-regulated in sulfur-grown cells; afe_0654 and afe_0674 were down-regulated, and the others were up-regulated in ferrous iron-grown cells. Afe_2126 was down-regulated, and the others were up-regulated when A. ferrooxidans DC exposed to Cd(2+). According to experimental results and bioinformatics analysis, the proteins encoded by afe_0654 and afe_0674 may relate with Mn(2+) and Cd(2+) efflux. It needed further study whether they relate with Zn(2+) transport. Proteins encoded by afe_0671, afe_1143, and afe_1144 may relate with the efflux of Mn(2+), Zn(2+), and Cd(2+). The protein encoded by afe_2126 may relate with Mn(2+) and Zn(2+) efflux and Cd(2+) uptake.
- MeSH
- Acidithiobacillus účinky léků genetika růst a vývoj MeSH
- bakteriální geny MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biologický transport MeSH
- hořčík metabolismus toxicita MeSH
- klonování DNA MeSH
- kovy metabolismus toxicita MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- mangan metabolismus toxicita MeSH
- mikrobiální testy citlivosti MeSH
- regulace genové exprese u bakterií účinky léků MeSH
- sekvenční analýza DNA MeSH
- síra metabolismus MeSH
- stanovení celkové genové exprese MeSH
- výpočetní biologie MeSH
- zinek metabolismus toxicita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- hořčík MeSH
- kovy MeSH
- mangan MeSH
- síra MeSH
- zinek MeSH
