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Microbiological Diagnosis of Fungal Keratitis
Published in Mahendra Rai, Marcelo Luís Occhiutto, Mycotic Keratitis, 2019
Species-specific probes might be used for the identification of the most important species of corneal pathogenic fungi. However, the range of fungi causing keratitis is significantly wide. Therefore, some species causing infection could remain unidentified by these molecular methods. The sequencing of ITS region allows this requirement. Small size of the DNA fragment permits its sequencing in both directions at once, and the obtained sequence gives enough information to identify the fungal species. Specific DNA microarray combining multiplex PCR and consecutive DNA chip hybridization to detect fungal genomic DNA in clinical samples other than ocular ones, was evaluated. This method can also be performed for ocular samples (Ferrer et al. 2001, Ghosh et al. 2007, Oechsler et al. 2009). Species-specific identification of a wide range of fungal pathogens can be performed by Luminex xMAP hybridization technology. This method is a kind of hybridization assay, which permits the analysis of up to 100 different target sequences in a single reaction vessel (Preuner and Lion 2013).
Respiratory Pathogens
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
Cloned viral DNAs probes are able to detect at least 5 pg of homologous DNA. An even higher sensitivity (3 pg of DNA) can be achieved in a dot-blot hybridization assay using in vitro synthesized, radioactively labeled single-stranded RNA (ssRNA) transcripts as probes.
Two-Dimensional Microfluidic Bioarray for Nucleic Acid Analysis
Published in Iniewski Krzysztof, Integrated Microsystems, 2017
In low-density DNA microarray analysis, the conventional pin-spotting method has been used to create dot-like probe arrays. The performance of the subsequent hybridization assay is thus heavily influenced by the quality of immobilized probe spots. In practice, because the spotting solutions are open to air and the spotting pins are very close to each other, the pin-spotting method may suffer from splashing, evaporation, and cross contamination [53]. In addition, probe spots of high homogeneity are beneficial, since they simplify image analysis and considerably enhance the accuracy of signal detection. When a spotting buffer such as saline sodium citrate solution is used, spot uniformity is often poor due to the hydrophobic properties of the chemically modified glass surface. Supplementary chemicals such as dimethyl sulfoxide can improve spot uniformity, but could introduce new problems such as spot size increase [54]. Moreover, during the blocking and cleaning procedures after spotting on the glass surface, the remaining unreacted probe molecules could diffuse away and smear the slide to form a comet-like spot [12]. Furthermore, if microchannel hybridization is going to be used later with a spotted microarray, additional devices such as steel clamps have to be used to ensure that the entire hybridization microchannel is well aligned to probe rows [52].
Strategies for the improvement of HCV testing and diagnosis
Published in Expert Review of Anti-infective Therapy, 2019
The gold standard for HCV genotype determination is the sequencing of a part of viral genome (mainly NS5B region or core region) followed by phylogenetic analysis. However, this method is time-consuming and cumbersome. Some laboratories use the line probe assay, a reverse hybridization assay using probes in both the 5ʹ non-coding and core regions of the viral genome. This assay discriminates well the six main HCV genotypes (1 to 6) and properly differentiates genotype 1 subtype 1a from 1b, thus fulfilling the clinical needs for treatment regimen decision. However, this assay has not been designed to accurately identify HCV subtypes other than 1a and 1b, nor to identify mixed infections emphasizing the need for a high-resolution system based on phylogenetic analysis of reads obtained by deep sequencing of a relevant genome region, as recently suggested [11,12]. Commercial assays based on phylogenetic analysis of reads obtained by deep sequencing of an NS5B fragment of the HCV genome would make it a method of choice for HCV genotyping and subtyping for future clinical practice and research [13].
Signalling pathways identified in salivary glands from primary Sjögren’s syndrome patients reveal enhanced adipose tissue development
Published in Autoimmunity, 2018
Lara A. Aqrawi, Janicke Liaaen Jensen, Gunnvor Øijordsbakken, Ann-Kristin Ruus, Ståle Nygård, Marit Holden, Roland Jonsson, Hilde Kanli Galtung, Kathrine Skarstein
The integrity of the total RNA samples was assessed using the Agilent 2200 Tape Station Nucleic Acid system. For each sample, 388 ng of total RNA was amplified and labelled using the Illumina Total Prep96 RNA Amp Kit (Thermo Fisher Scientific, Waltham, MA), following the manufacturer’s protocol. The quantity of the labelled complementary RNA (cRNA) was measured using the NanoDrop Spectrophotometer, and the quality and size distribution of the labelled cRNA was assessed using the 2200 Tape Station. For each sample, 750 ng of biotin labelled cRNA was hybridized to Illumina’s HumanHT-12 v4 Expression BeadChip. The hybridization, staining, and washing were conducted using Whole-Genome Gene Expression Direct Hybridization Assay (Illumina, San Diego, CA), according to the manufacturer’s protocol. Raw data were imported to Illumina’s GenomeStudio software V2011.1, Gene Expression module v. 1.9.0, and text files with both non-normalized and quantile normalized data were exported. The intactness of the biological specimen was monitored by Housekeeping gene controls, consisting of two probes per housekeeping gene that should be expressed in any cellular sample (Supplementary Table 1).
Adrenal-dependent and -independent stress-induced Per1 mRNA in hypothalamic paraventricular nucleus and prefrontal cortex of male and female rats
Published in Stress, 2018
Lauren E. Chun, Jenny Christensen, Elizabeth R. Woodruff, Sarah J. Morton, Laura R. Hinds, Robert L. Spencer
In situ hybridization for Per1, Per2, Bmal1, and cFos mRNA followed procedures previously reported (Chun et al., 2015). Briefly, sections on slides were fixed with 4% paraformaldehyde, went through a series of standard saline citrate (SSC) solution washes, then bathed in triethanolamine and acetic anhydride solution. Sections were dehydrated in increasing concentrations of ethanol baths before being air dried. Hybridization buffer containing the 35S-labelled riboprobe for each gene of interest was applied to each slide. Hybridization occurred in humidified chambers with 50% formamide and 50% water for 16–20 h. Coverslips were gently removed in SSC baths. Tissue was then exposed to 0.02 g/L RNase at 37 °C, washed in decreasing concentrations of SSC, and incubated in SSC at 65 °C for 1 h. Sections were dehydrated in increasing concentrations of ethanol, then air dried. Slides were set on Kodak BioFilm Maximum Resolution Autoradiography Film (Carestream Health, Windsor, CO) for 2–4 weeks, then developed in a medical film processor SRX-101A (Konica Minolta, Tokyo, Japan). Each in situ hybridization assay was separated by experiment and by region of interest (ROI; SCN and PVN were run together).