1,720,957 research outputs found
Development of a rapid molecular method for detection of C. perfringens and other pathogens in water sources
No full textAccess to clean water is a basic requirement for all. Global water scarcity necessitates the reclamation and reuse of water. Effective, rapid surveillance for pathogens in recycled water is essential, assisting pathogen detection in water intended for reuse by humans, animals, and industry. Monitoring of repurposed water is essential as it might contain residual microorganisms that are less sensitive to disinfection. Examples are Clostridium perfringens (a spore forming microorganism with a cell envelope that is less sensitive to chemical disinfection and the cause of diarrhoea via contaminated water and food) and Legionella pneumophila (a waterborne, pulmonary pathogen protected from disinfection due to its intracellular lifestyle inside Protists such as Entamoeba). Molecular detection of nucleic acids is a tool enabling more rapid detection of pathogens in water compared to culture-based methods. Molecular tests using nucleic amplification and detection via the polymerase chain reaction (PCR) method needs thermal cycling instruments and thus is limited in resource-poor environments and spaces lacking infrastructure such as electricity. A solution is to use isothermal amplification methods, requiring heated devices at a single temperature and excluding the need for thermal cycling devices. A battery-operated heated device suffices for isothermal methods such as Recombinase Polymerase Amplification (RPA) and requires less time compared to PCR. Furthermore, isothermal amplification, when combined with the detection of generated amplicons on lateral flow devices, excludes the need for detection methods requiring electrified equipment. The study described here aimed to evaluate RPA, combined with lateral flow detection of labelled amplicons, as a tool to detect deoxyribonucleic acid (DNA) from two waterborne pathogens, i.e., Clostridium perfringens (C. perfringens) and Legionella pneumophila (L.pneumophila). In-house designed primers targeting the C. perfringens phospholipase C gene (plc) and previously published primers to detect L. pneumophila mipA gene were evaluated using conventional PCR and RPA. The lowest amount of DNA needed for amplification and detection was determined using PCR and both agarose gel electrophoreses and lateral flow detection (LFD). For LFD, labelled primers were used to generate dual-labelled amplicons that were captured and detected on a commercial lateral flow device. The RPA-LFD method was then tested with DNA extracted from a limited number of water samples provided by the local municipality. Lastly, as efficient DNA extraction is a limiting step in molecular detection. Consequently, three commercial, manual DNA purification methods were evaluated for their ability to extract DNA from C. perfringens. The generated data showed successful detection of DNA from both pathogens via RPA-LFD). A minimum of at least 1x10-3 ng of pure DNA, extracted from the cultured pathogens, were efficiently amplified, and detected for both microorganisms. Future work should evaluate the method more extensively with DNA extracted from variety of real-world water samples as PCR inhibitors in water from different resources might influence the sensitivity of the method. Regarding DNA extractions, we compared three different DNA extraction and purification methods (solid phase, desalting and magnetic bead purification) to obtain fit for purpose DNA from cultured C. perfringens. Extraction kits were also compared for equipment and infrastructure needed, efficiency of extraction, cost, and ease of use. All kits generated DNA fit for downstream amplification, but consideration should be given to kits that require fewer steps to obtain pure DNA and use minimal equipment. Kits with a removal step for PCR inhibitors in water should also preferentially be used. In conclusion, the data shows that RPA-LFD could be a feasible method to monitor pathogens in water. Further development of the RPA-LFD method using dual labelled amplicons are necessary, including validation of the method with an expanded set of real-world water samples. The method should further be combined with viability testing, to confirm that DNA detected from samples originates from viable microorganisms and not cell free DNA present in water
Molecular detection and study of Campylobacter and related microorganisms
Get PDFPhilosophiae Doctor - PhDSpecies of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals; and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42ºC as well as at ambient room temperature (±222ºC). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4ºC for up to 297 days.South Afric
Phenotypic and molecular analysis of Helicobacter spp. and related micro-organisms identified in clinical & environmental specimens
No full textIncludes bibliographical references (p. 140-168)
Molecular detection and study of Campylobacter and related microorganisms
No full textPhilosophiae Doctor - PhDSpecies of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals; and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42ºC as well as at ambient room temperature (±222ºC). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4ºC for up to 297 days.South Afric
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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