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Bacterial Communities in the Pathogenesis of Necrotizing Enterocolitis
Published in David J. Hackam, Necrotizing Enterocolitis, 2021
Brigida Rusconi, Misty Good, Barbara Warner
The requirement for a dysbiotic community for NEC to occur has been well established in both human and animal model studies (20). The central role of the microbiota in NEC development is best illustrated by the pioneering studies done by Musemeche et al. in germ-free rats (21) and Sangild et al. in preterm piglets (22). These studies complemented by results obtained in mice (23, 24) and quail (25) clearly show a reduced incidence of NEC-like lesions in the absence of a microbial component. In humans, a role for bacteria in NEC has long been proposed, supported by the timing of onset occurring after gut microbial colonization (26) and reports of sporadic NEC epidemics in NICUs, with or without specific organisms implicated in the etiology (27–31). To investigate the presence or absence of many bacterial species within the same sample, 16S rDNA-denaturing gradient gel electrophoresis (DGGE) can be used. When DGGE was applied by Smith et al. on a cohort of preterm infants, the NEC cases could not be distinguished from matched controls based on the presence or absence of bacteria alone (32). These data suggest that NEC is not being driven by specific bacterial species. Nevertheless, bacteria still play an essential role in NEC, and with the advent of large-scale 16S sequencing, investigators have been able to look at changes in proportions and composition of the microbial community in much more detail.
Malassezia
Published in Rossana de Aguiar Cordeiro, Pocket Guide to Mycological Diagnosis, 2019
Reginaldo Gonçalves de Lima-Neto, Danielle Patrícia Cerqueira Macêdo, Ana Maria Rabelo de Carvalho, Carolina Maria da Silva, Rejane Pereira Neves
As previously mentioned, proper identification of Malassezia species can be attained by molecular methods. Many molecular tools have been established for Malassezia identification and also for the detection of intraspecific genetic variation: (a) direct sequencing of rDNA loci (internal transcribed spacers ITS-1 and ITS-2, intergenic spacer IGS-1, large subunit LSU), chitin synthase gene and RNA polymerase subunit 1 gene, (b) PCR-based restriction fragment length polymorphism (PCR-RFLP) of rDNA loci, (c) random amplification of polymorphic DNA (RAPD), (d) denaturing gradient gel electrophoresis (DGGE), and (e) pulsed field gel electrophoresis (PFGE) (Cafarchia et al., 2011). Molecular methods suitable for identification of Malassezia species in clinical mycological laboratories are described in Table 6.3.
Molecular Genetics and Diagnostic Testing
Published in Merlin G. Butler, F. John Meaney, Genetics of Developmental Disabilities, 2019
Searching for disease-causing mutations in a gene may require identification of a single nucleotide or a few nucleotides in a coding sequence composed of thousands of nucleotides. To avoid sequencing large amounts of DNA, strategies have been developed to rapidly scan genes for small mutations. The techniques use PCR to amplify the gene or gene segment to be scanned and, ultimately, each method leads to sequencing of the region shown to contain a mutation. Single-strand conformational polymorphism analysis is based on the conformational change in DNA resulting in an alteration in electrophoretic mobility that is produced by some mutations.Mismatch heteroduplex analysis is a method in which the PCR products are denatured and allowed to renature forming heteroduplexes, if a mismatch (mutation) is present. The heteroduplexes can be distinguished because they move more slowly on nondenaturing polyacrylamide gels than do homoduplexes.Denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography are methods that are based on the differences in mobility of heteroduplex and homoduplex molecules when they are heated to temperatures just below the melting temperature of the homoduplexes.
Beverages Rich in Resveratrol and Physical Activity Attenuate Metabolic Changes Induced by High-Fat Diet
Published in Journal of the American College of Nutrition, 2021
Letícia Monteiro da Fonseca Cardoso, Juliana Arruda de Souza Monnerat, Isabelle Waleska Santos de Medeiros Silva, Raiza da Silva Ferreira Fiochi, Nina da Matta Alvarez Pimenta, Bruna Ferreira Mota, Manuela Dolisnky, Flavia Lima do Carmo, Sergio Girão Barroso, Carlos Alberto Soares da Costa, Milena Barcza Stockler-Pinto, Gabrielle de Souza Rocha
Denaturing gradient gel electrophoresis (DGGE) of the amplified extracted, DNA was performed using a DCode system universal mutation detection system Bio-Rad® (VA, USA). The products were analyzed to be applied directly to the gel containing 6% (w/v) polyacrylamide and 0.5 x TAE (20 mM Tris-acetate [pH 7.4], 10 mM sodium acetate, 0.5 mM EDTA). The gel contained 6% acrylamide with a gradient of 45–65% denaturant (urea and formamide). The electrophoresis time was 16 h at 60 °C and 75 V. After electrophoresis, the gel was stained for 40 min with SYBR Gold I (Molecular Probes, Inc., OR, USA) and scanned in image capture system STORM™ 860 Imaging System (GE Healthcare, USA). Results were presented as dendrograms constructed after image capture and analysis by Pearson correlation coefficients (r). Cluster analysis was performed by the unweighted pair group method with average linkages (UPGMA) using BioNumerics Software (Applied Math, Belgium) (20).
Innovative application of nested PCR for detection of Porphyromonas gingivalis in human highly calcified atherothrombotic plaques
Published in Journal of Oral Microbiology, 2020
Adrian Brun, Hélène Rangé, Bastien Prouvost, Mikael Mazighi, Yvonne Kapila, Philippe Bouchard, Jean-Baptiste Michel
Nested PCR is a modification of the conventional PCR method, which consists of a succession of two PCRs, the second PCR uses the product of the first PCR as sample. Indeed, two sets of primers are used in the nested PCR protocol. The first set of primers (first PCR) amplifies large fragments by binding outside of the target DNA. The second set of primers (second PCR) binds specifically at the target DNA. It is intended to reduce non-specific binding by reducing the amplification of unexpected primer binding sites. Different methods have been described for purifying the PCR products from the first universal PCR (using 16S rRNA gene universal primers). One option is the Denaturing Gradient Gel Electrophoresis (DGGE) technique, which involves using a gel extraction kit after the amplified products have migrated on an agarose gel. Alternative options include using PCR purification kits or incubation with an enzyme that removes unincorporated primers and dNTPs. Use of the Illustra ExoProStar® (Dutcher) product for removing unincorporated primers and dNTPs is easier to implement compared to other methods, and was highly effective in our study.
Efficacy of the National Thalassaemia and Sickle Cell Disease Prevention Programme in Northern Greece: 15-Year Experience, Practice and Policy Gaps for Natives and Migrants
Published in Hemoglobin, 2018
Stamatia Theodoridou, Nikolaos Prapas, Angeliki Balassopoulou, Effrossyni Boutou, Timoleon-Achilleas Vyzantiadis, Despoina Adamidou, Evangelia-Eleni Delaki, Eleni Yfanti, Marina Economou, Αikaterini Teli, Olga Karakasidou, Evangelia Skatharoudi, Theodoros Theodoridis, Ersi Voskaridou
The screening methods used for DNA analysis were denaturating gradient gel electrophoresis (DGGE), allele-specific oligonucleotide (ASO) analysis, high resolution melting analysis (HRMA) and other polymerase chain reaction (PCR) directed methods [amplification refractory mutation system (ARMS), gap-PCR (PCR amplification across breakpoints)] and DNA sequencing. In selected cases, Hb biosynthesis was performed at the Molecular Genetics Laboratory of the National Thalassaemia Centre, Laiko General Hospital of Athens, Athens, Greece.