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PROBE FOR THE DETECTION OF BACTERIAL INFECTIONS
No full textThe present invention relates to a nucleotide probe specific for Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis, its uses, and methods for detecting said microorganisms in a sample of interest, and kits comprising said probe and suitable reagents
METHOD AND SYSTEM FOR DETERMINING THE PRESENCE AND/OR AMOUNT OF AT LEAST ONE ANALYTE THAT MAY BE CONTAINED IN A SAMPLE
No full textThe present invention relates to a method for determining the presence and/or amount of at least one analyte (T) that may be contained in a sample. The method comprises: (a) a recognition step during which said sample (E) is placed in the presence of at least one primary probe (1), in order to form, if necessary, a primary complex (C1), (b) a development step during which at least one developing reagent is added, capable of forming, starting from the primary probe (1) of said primary complex (C1), a secondary complex (C2), said at least one developing reagent comprising: - at least one amplification probe (3), of the core (31) - shell (32) type, wherein said shell (32) comprises several molecules of an affinity tag (321), and - a tracer (21) for converting the secondary complex (C2) into a visible and/or measurable signal, said tracer (21) being bound or intended to bind with the affinity tag (321) of said at least one amplification probe (3), so that in said secondary complex (C2) at least some of the molecules of said affinity tag (321) of said at least one amplification probe (3) are bound with said tracer (21)
DEVELOPMENT OF AN ELECTROCHEMICAL BIOSENSOR FOR FOOD SAFETY: DETECTION OF FOOD PATHOGENS
Get PDFL. monocytogenes and Campylobacter spp. are two important foodborne pathogens.
They can be acquired by ingestion of contaminated food mainly ready to eat (RTE), undercooked chicken, and milk and dairy products respectively. The symptoms are gastrointestinal disorders that can switch in a serious disease like listeriosis and campylobacteriosis in weak individuals.
The currently recommended ISO standard methods are sensitive and ensure compliance with microbiological criteria, but require long times and a lot of work.
In order to avoid recalls or economic losses, food industries need rapid protocols that can provide results in short times.
In this thesis, with the perspective to develop rapid and efficient molecular detection methods, species-specific primers and probes were designed for L. monocytogenes and C. jejuni, C. coli, C. lari, and C. upsaliensis.
For the detection of L. monocytogenes in cold-smoked salmon samples and ham factory samples designed MAR1- MARB primers were applied in PCR and qPCR protocols.
Two probes, ListCapt and ListE, were tested for the detection of Listeria monocytogenes. The ListCapt probe was applied on a DNA-biosensor based on the organic electrochemical transistor (OECT), after preliminary optimization tests with dot blot assay. Instead, the ListE probe was applied on a DNA- electrochemical biosensor based on voltammetry.
Samples of cold-smoked salmon (CSS) and Ham factories samples (from food and environment) were analyzed by electrochemical biosensors. In parallel to molecular methods, AFNOR validated Listeria PrecisTM method (ISO standard equivalent method) was applied to the food samples, to compare traditional plate count methods to molecular techniques, in both traditional and molecular approaches. L. monocytogenes was detected in only one of the tested CSS samples. Instead, six samples from ham factories were positive to the presence of L. monocytogenes. Both PCR and qPCR protocols allowed the detection of L. monocytogenes, confirming the capability of primers to detect the pathogen from a complex matrix. However, an enrichment step of 24 h was necessary.
After dot blot protocol optimization to assess specificity and sensitivity of oligonucleotide probes were employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. The studies have been made with promising results, anticipating the prospective potential of the system for label-free DNA sensing.
For the detection of Campylobacter spp. designed primers CampyP were applied in PCR and qPCR protocols in 20 chicken meat samples. The probe CampyP3 was tested for the detection of Campylobacter spp. and was applied to a DNA-electrochemical biosensor based on voltammetry.
Chicken meat samples were analyzed by the electrochemical biosensor. In parallel to molecular method, ISO 10272-1:2006 was applied to food samples, to compare traditional plate count methods to molecular techniques. With both traditional and molecular approaches, Campylobacter was detected in five tested samples.
Both PCR and qPCR protocols allowed the detection of Campylobacter spp., confirming the capability of primers to detect the pathogen matrix without enrichment for the application of qPCR technique. After dot blot protocol optimization, to assess specificity and sensitivity CampyP3 was employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. After the optimization, some food samples were analyzed and confirmed the data obtained by ISO and qPCR
Development and application of molecular probes
No full textThe development of DNA probes started from 1950's for diagnostic purposes and it is still
16 growing. DNA probes are applied in several fields such as food, medical, veterinary, environment
17 and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits
18 microorganism identification, including pathogen detection, and their quantification when used in
19 specific systems. Various techniques obtained success by the utilization of specific DNA probes, that
20 allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that
21 were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon
22 resonance (SPR), electrochemical, and quartz crystal microbalance (QCM) biosensors that use
23 different transduction systems
From vineyard to wine, Lactic Acid Bacteria and yeast identification using molecular methods
Get PDFUsually all attempts to characterize the microbial diversity in wine fermentations have
employed standard methods of enrichment and isolation to cultivate various microbial
constituents before taxonomic identification. This estimation of microbial diversity, in
addition to being time-consuming, is often problematic since many microorganisms may not
grow on standard laboratory media. Moreover culture-independent molecular methods allow
more rapid profiling of complex populations, or quantification of targeted species, thereby
enhancing the information available to the winemaker.
The aim of the present study is to describe the yeast and lactic acid bacteria communities
found in the vineyard, the winery and the wine, using culture-dependent and culture
independent molecular methods. Samples came from botrytized Picolit grapes during 2013
vintage in Corno di Rosazzo, Friuli Venezia Giulia Region, Italy.
On this work, the DNA extracted directly from the must and wine (culture-independent
technique) was analyzed by denaturing gradient gel electrophoresis (DGGE), as well as the
DNA extracted from isolated colonies that came from the vineyard and winery samples
(culture-dependent technique). Both types of DNAs were specifically amplified by PCR using
particular groups of universal primers depending on the nature of the sample (bacteria,
Saccharomyces or non-Saccharomyces yeasts).
Saccharomyces sensu stricto yeasts were amplified with ShafGC and Shar specific primers.
This yeast was found in the Winery and in the Picolit wine. The DGGE study showed that all
samples corresponded to the Saccharomyces cerevisiae species, being identical to the
commercial yeast used as a starter.
Non-Saccharomyces 26S ribosomal rDNA genes were amplified by Nested PCR using the
primers NL1-NL4 for step 1 and NL1GC-LS2 for step 2. In the vineyard, the DGGE analysis
allowed the identification of several yeasts like Kloeckera sp., Metchnikowia sp., Pichia sp.,
Hansenula sp. and Schizosaccharomyces sp. Other than Saccharomyces cerevisiae, no other yeast was found in the wine, indicating the starter’s ability to reduce the variability of the
yeasts during the fermentation process. Brettanomyces sp. and Candida sp. were absent in the
yeast analyzed. Samples that didn't correspond to a reference strain used were sequenced
allowing the identification of Torulaspora delbrueckii and Debaryomyces hansenii.
Finally, bacteria samples were analyzed by employing gradient gel electrophoresis (DGGE) of
polymerase chain reaction (PCR) amplified with 338fGC and P4V3 primers. Several
microorganisms were found in the vineyard and the winery but only the strains of
Lactobacillus casei/ Lactobacillus paracasei and Leuconostoc mesenteroides were found in
the Picolit wine, indicating the selection due to the yeast fermentation process. Only 2 samples
of the vineyard and one contact plate of the cellar were identified as Oenococcus oeni.
Samples that didn't correspond to a reference strain used were sequenced allowing the
identification of Lactobacillus hilgardii, Lactobacillus mali, Pediococcus parvulus.
In this work we demonstrate that PCR-DGGE is a viable alternative to standard plating
methods for a qualitative assessment of the microbial constituents from de vineyard to the
wine.Fil: Varela, Anabella. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias
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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