Diagnosis of Chagasic Encephalitis by Sequencing of 28S rRNA Gene.
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ABSTRACT:
SUBMITTER: Multani A
PROVIDER: S-EPMC6590746 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA barcoding is an important molecular methodology for species identification that was developed over the last two decades and it should be covered in the biology bachelor curriculum. Here, we present an example of DNA barcoding by sequencing a segment of the 28S nuclear ribosomal large subunit rRNA gene of wild mushrooms and framing the education in a project form for undergraduate students in biology. Students perform this project in 6-8 weeks, which also includes preparing a poster, writing a report and presenting a paper related to the work in a journal club format. First, fieldwork in the Netherlands was carried out, during which students collected mushrooms under supervision of a professional mycologist with the goal to (a) verify morphologically based identifications with a molecular method and (b) assess phylogenetic relationships of the different species collected. Next, DNA extractions and quantitation were performed, PCR amplification was done, and samples were sent out for Sanger sequencing. Students aligned and analyzed the sequences using BLAST and Geneious and subsequently created a phylogenetic tree. In case of collecting DNA barcodes of an earlier sequenced species, students could upload the data to a repository established for facilitation of future research projects. The method described is very robust, reagents and equipment are readily available, and costs are relatively low. In addition, the results can be compared to published fungal phylogenetic trees.
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Project description:Fusarium spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against Fusarium isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33 Fusarium isolates belonging to six Fusarium species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera Acremonium and Cylindrocarpon. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of Fusarium species isolated from patients.
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Project description:BACKGROUND:Loricifera is an enigmatic metazoan phylum; its morphology appeared to place it with Priapulida and Kinorhyncha in the group Scalidophora which, along with Nematoida (Nematoda and Nematomorpha), comprised the group Cycloneuralia. Scarce molecular data have suggested an alternative phylogenetic hypothesis, that the phylum Loricifera is a sister taxon to Nematomorpha, although the actual phylogenetic position of the phylum remains unclear. METHODS:Ecdysozoan phylogeny was reconstructed through maximum-likelihood (ML) and Bayesian inference (BI) analyses of nuclear 18S and 28S rRNA gene sequences from 60 species representing all eight ecdysozoan phyla, and including a newly collected loriciferan species. RESULTS:Ecdysozoa comprised two clades with high support values in both the ML and BI trees. One consisted of Priapulida and Kinorhyncha, and the other of Loricifera, Nematoida, and Panarthropoda (Tardigrada, Onychophora, and Arthropoda). The relationships between Loricifera, Nematoida, and Panarthropoda were not well resolved. CONCLUSIONS:Loricifera appears to be closely related to Nematoida and Panarthropoda, rather than grouping with Priapulida and Kinorhyncha, as had been suggested by previous studies. Thus, both Scalidophora and Cycloneuralia are a polyphyletic or paraphyletic groups. In addition, Loricifera and Nematomorpha did not emerge as sister groups.
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Project description:Lymphatic filariasis is one of the major public health concern in India, and Bankura district of West Bengal is one of the main filaria prone area of the country. Wuchereria bancrofti is the causative organism and Culex quinquefasciatus is the main vector of lymphatic filariasis in India. In the present study, infection and infectivity rate of filarial vector C. quinquefasciatus were determined. The molecular characterization, DNA fingerprinting and phylogenetic analysis of W. bancrofti was done. In the study area, overall vector infection and infectivity rates were 4.83 and 0.97%, respectively. The infection and infectivity rate were found to be higher in rainy season and lower in summer season. The AT and GC content of W. bancrofti SNC Bankura (Accession No. JF930705) were 55.59 and 44.41%, respectively. The phylogenetic tree was prepared following neighbour joining, maximum parsimony, minimum evolutionary and UPGMA methods. The study revealed that W. bancrofti SNC Bankura (JF930705) showed 100% similarity with W. bancrofti (Accession No. EU370161). The cluster containing, W. bancrofti SNC Bankura (JF930705) and W. bancrofti (EU370161) branched with Brugia pahangi (M15309) with 62% bootstrap value. W. bancrofti SNC Bankura (JF930705), W. bancrofti (EU370161) and B. pahangi (M15309) branched with Dirofilaria immitis (AF188120) with 78% bootstrap value.
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Project description:The RNA methyltransferase NSUN5 modulates the lifespans of yeast, worms and flies by re-programming the translation of specific subsets of mRNAs, which are associated with the cellular stress response. Here we show by RNA bisulfite sequencing that Rcm1 specifically methylates C2278 of yeast 25S rRNA and nsun-5 C3381 of C. elegans 28S rRNA.
2014-11-10 | GSE63113 | GEO
Project description:BackgroundMetagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test.MethodsIn a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review.ResultsWe enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment.ConclusionsRoutine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).
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Project description:Fasciolosis is a parasitic disease of medical and economic importance. This retrospective study was conducted on 110 Fasciola flukes collected from livers of 14 infected Naimi sheep slaughtered at Riyadh abattoir in Saudi Arabia during winter season of 2016. Collected specimens were analyzed for their species identification on the basis of partial sequences of mitochondrial 28S rRNA gene. Results have shown the presence of both Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica) species. Where Fasciola hepatica was predominate (80%). Both intra-species and interspecies genetic distance was studied and results showed that the intraspecific variability among individuals of both species i.e., F. hepatica and F. gigantica, ranging between 0 and 1% while the interspecific diversity between F. hepatica and F. gigantica was only 1%. In conclusion, mitochondrial 28S rRNA gene is a proved as a good marker in identifying Fasciola of different species. Where, the F. hepatica and F. gigantica are present in sheep breed in Riyadh region, Saudi Arabia.
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