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Comparação entre as técnicas de RT-PCR e inoculação intracerebral em camundongos para detecção do vírus da raiva em amostras mantidas por longos períodos em diferentes estados de conservação /Lopes, Marissol Cardoso. January 2008 (has links)
Orientador: Luzia Helena Queiroz da Silva / Banca: Avelino Albas / Banca: Silvana Regina Favaretto Lazarini / Resumo: As análises antigênica e genética são ferramentas importantes para o estudo da epidemiologia da raiva em uma região. A recuperação e reisolamento viral a partir de amostras conservadas por longos períodos em temperatura de congelamento é essencial para estudos retrospectivos. Porém, o tempo de conservação, associado a repetidos ciclos de congelamento e descongelamento, promove uma perda significativa na viabilidade do vírus, condição esta que pode ser contornada com a utilização de técnicas de biologia molecular, como a RT-PCR. Com o objetivo de verificar a viabilidade e detectar o RNA do vírus rábico, 95 amostras com diagnóstico positivo e armazenadas por 4 a 13 anos a -20 e -80ºC foram avaliadas por inoculação intracerebral em camundongos e RT-PCR. Apenas 33,6% (32/95) das amostras inoculadas em camundongos foram positivas, enquanto que a RTPCR detectou o genoma viral em 65,3% (62/95). Houve diferença estatisticamente significativa (p<0,0001) na viabilidade das amostras e na detecção do genoma viral na amostras armazenadas por mais de 10 anos, sendo a porcentagem de positividade de 22,1% e 59,7%, respectivamente. O presente estudo confirma a importância da RT-PCR na detecção do genoma viral em amostras conservadas por longo período de tempo, incluindo aquelas em estado visível de decomposição. / Abstract: The antigenic and genetic analyses are important tools for retrospective study of rabies epidemiology in a region. The recovery and viral re-isolation from samples conserved for long periods in freezing temperature are essential for these studies. However, time conservation, associated with temperature variations, causes a significative virus viability loss. On the other hand, molecular tools, such as RT-PCR, can overcome this condition. For this purpose, 95 positive samples stored for 4 to 13 years at -20 and -80ºC were evaluated by intracerebral inoculation in mice and RT-PCR. Of this total, only 33,6% (32/95) had been positive in the intracerebral inoculation, while RT-PCR detected the viral genoma in 65.3% (62/95). It had a significant difference (p>0,0001) in the viability of the samples and the detention of the viral genoma from those samples store for more than 10 years and the percentage of positivity reached 22,1% and 59,7%, respectively. The present study confirms the importance of the RT-PCR technique for detection of viral genoma in old samples, including those in apparent state of decomposition. / Mestre
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Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)Pillay, Sarveshni January 2007 (has links)
Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.
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Molecular diagnostic approach to determine the degree of photoaging of the skinWilcox, Stephany Vanessa 04 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Context: Excessive exposure to ultraviolet radiation (UV) results in the risk of acquiring long-term harmful
effects such as photoaging, which is characterised by deep wrinkles, roughness, dyspigmentation and an
increased loss in elasticity. As a result, the detection of photoaging at an early stage is crucial to improving
morbidity, whilst preventing the advancement of skin cancer.
Aim: The aim of the study was to develop and to validate a diagnostic real-time PCR method in order to
establish the gene expression profiles of potential biomarkers in the skin so as to quantify the degree of
photoaging: this was conducted by retrieving total RNA from cells adherent to tape strips from sun exposed
and non-exposed skin areas.
Materials and methods: Twenty healthy volunteers consisting of seven males and thirteen females aged
25 to 67 years were included in this study. Tape stripping was performed using pre-cut D-Squame® 22 mm
adhesive discs. Samples were collected on the right medial thigh area 20 cm above the patella and 2 cm
below the lateral canthus of the right eye. Total RNA was extracted and relative standard curve method of
gene expression was performed. TGF-β, MMP 9, TNF-α and IL-6 mRNA transcripts were selected as
representative cytokines to determine the relative fold-change in sun exposed and non-exposed areas of the
skin so as to determine extent of photoaging.
Results: Repeatability and reproducibility was determined by the coefficient of variation (CV) was within an
acceptable range. Thirty five percent (n=7) samples displayed down-regulatory effects for TGF-β. Down
regulation of MMP 9 was observed within 30% (n=6) of samples, while 15% (n=3) showed marked up
regulation. Only two samples showed measurable levels of TNF-α in the assay, of which one showed
significant up regulation. Furthermore, we were unable to detect any IL-6 expression in any of the samples
prepared.
Conclusion: we have shown that epidermal cytokines can be retrieved from tape stripped samples and can
be quantified via real-time PCR. However, the choices of cytokine biomarkers reveal that they are as
important as the concentration of starting material. In this study cytokines such as IL-6 is not as informative
in determining the extent of photoaging without high doses of ultraviolet radiation before sample collection
as opposed to the other explored cytokines. / AFRIKAANSE OPSOMMING: Konteks: Oormatige blootstelling aan ultraviolet (UV) bestraling kan tot ‘n risiko van skadelike en
lantermynse nagevolge lei wat gekenmerk word deur foto-veroudering. Dit sluit in diep plooie, growwe vel
en ‘n toenemende verlies in elastisiteit. Die ontdekking van foto-veroudering op ‘n vroeë stadium is van
kardinale belang vir die verbetering van morbiditeit en die voorkoming van velkanker bevordering.
Doelstelling: Die doel van hierdie studie was om ‘n diagnostiese polimerase kettings reaksie (PKR) metode
te ontwikkel om geen uitdrukkings profiele van potensiële bio-merkers te vestig in die vel, om so die graad
van foto-veroudering in areas van vel wat blootgestel word aan die son en beskermde van die son te bepaal
deur totale RNS te versamel van kleeflintskyfies.
Materiale en metodes: Twintig gesonde vrywilligers (sewe mans en dertien vroue), tussen die ouderdom
van 25 en 67 jaar, was ingesluit in hiedie studie. Vel monsters was versamel deur gebruik te maak van Dsquame®
22 mm kleeflintskyfies 20 cm bokant die patella van die regterkanste mediale heup en 2 cm onder
die regter oog. Totale RNS was geisoleer en die relatiewe vlak van geen uitdrukking was bepaal deur
gebruik te maak van die kurwe model. Die boodskapper ribonukleiosier transkripsies van die sitokiene TGF-
β, MMP 9, TNF-α en IL-6 was gekies as verteenwoordigers van foto-veroudering om die relatiewe
verandering van foto-veroudering in die vel te bepaal.
Resultate: Validering metodes was aanvaarbaar. ‘n Afwaarts reguleringseffek in TGF-β en MMP 9 merker
uitdrukking is gevind in vyf en dertig persent (n=7) en dertig persent (n=6) van monsters, onderskuidelik. In
vyftien persent (n=3) van monsters is ‘n opwaarts reguleringseffek in die laasgenoemde gevind. Slegs twee
monsters het meetbare vlakke van TNF-α getoon in die eksperiment, waarvan slegs een ‘n
noemenswaardige opwaartse regulering getoon het. IL-6 uitdrukking is nie gevind in enige van die
monsters.
Gevolgtrekkings: Hierdie studie het bepaal dat sitokiene van die vel geisoleer van kleeflint monsters en
gekwantifiseer deer relatiewe PKR uitdrukking bepaal kan word. Die keuse van bio-merkers is egter net so
belangrik as konsentrasie bepaling van die monsters. Die IL-6 sitokien, in vergelyking met ander, is slegs
informaliet tydens hoë ultraviolet bestraling aan die vel blootgestel is.
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PCR detection, denaturing gradient gel electrophoresis (DGGE) fingerprinting and identification of the microbial consortium in different types of UASB granulesKeyser, Maricel 12 1900 (has links)
Thesis (PhD (Food Science))--University of Stellenbosch, 2006. / High-rate anaerobic bioreactors are used for the treatment of various wastewaters, of which the upflow anaerobic sludge blanket (UASB) bioreactor has the widest application, especially in the food and beverage industries. In an UASB bioreactor sludge develops in a particular granular or flocculent form and the success of the anaerobic process relies on the formation of active and settable granules. These granules are formed by self-aggregation of bacteria that can be divided into different trophic groups that are responsible for the metabolic breakdown of organic substrates.
The successful performance of a bioreactor is influenced by the composition of the substrate which subsequently may have an impact on the microbial consortium present in the UASB granules. In order to determine if a change in the structure of the non-methanogenic microbial community takes place, UASB brewery granules were subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium (5 g.l-1). No changes in the microbial community were observed when the granules were stressed with glucose medium as carbon source, regardless of an increase in the glucose concentration.
In order to better understand the effect that different wastewaters may have on the microbial consortium present in different UASB granules, the polymerase chain reaction (PCR) based denaturing gradient gel electrophoresis (DGGE) technique and sequence analysis were used to fingerprint and identify the Bacteria and Archaea present in either, winery, brewery, distillery or peach-lye canning UASB granules. Each granule type showed distinct PCR-based DGGE fingerprints with unique bands, while other bands were found to be present in all the granules regardless of the wastewater being treated. Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes, Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas, Nitrospira, Synergistes, Rhodococcus, Rhodocyclus, Syntrophobacter and uncultured bacteria were identified, representing different acidogenic, acetogenic and homoacetogenic Bacteria.Different methanogenic bacteria such as Methanosaeta, Methanosarcina, Methanobacterium and uncultured bacteria belonging to the group Archaea were also fingerprinted and identified from different UASB granules. In both these studies a DGGE marker was constructed that may be used to assist in the identification of bacteria. The DGGE marker can also be used to monitor the presence of bacteria over a time period during anaerobic digestion. Bioaugmentation or the enrichment of granules results in tailor-made granules that may be used for the treatment of specific wastewaters.
One of the most important contributions to the maintenance and enhancement of UASB granule formation is the inclusion of suitable microbes in the granule structure. Enterobacter sakazakii was isolated from raw winery wastewater and was found to produce sufficient amounts of desired fatty acids. This bacteria was, therefore, incorporated into batch cultured granular sludge. In order to identify and monitor the presence of the incorporated E. sakazakii in the tailor-made granules, 16S rRNA gene sequence primers and PCR conditions were developed.
The use of molecular techniques such as PCR-based DGGE and sequence analysis proved to be successful methods to fingerprint and identify the microbial consortium present in the different UASB granules.
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Genetic investigations of pneumocystis jirovecii : detection, cotrimoxazole resistance and population structureRobberts, Frans Jacob Lourens 12 1900 (has links)
Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / Pneumocystis jirovecii is a significant contributor to the burden of disease in
immunocompromised patients. The polymerase chain reaction (PCR) is more
sensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough and
treatment failures have been reported, and associated with mutations at codons 55
and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic or
population genetic models have been successful in elucidating P. jirovecii
intraspecies strain relatedness.
Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescence
microscopy (IF); 2) Determine the extent of co-infecting pathogens associated with
Pneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNA
strain types, and model lineage evolution employing a coalescent-theory based
statistical parsimony network analysis; 4) Investigate the possible emergence of
cotrimoxazole-resistant strains
Methods: PCR was evaluated on clinical specimens employing: ITS nested; DHPS
single and nested; DHFR nested; major surface glycoprotein (MSG) heminested;
mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetube
nested, and real-time 5S rRNA PCR. Retrospective analysis of co-infecting
pathogens seen in PcP patients was conducted. ITS regions were amplified, cloned
and sequenced. Statistical parsimony was applied for coalescence based network
genotype analysis. DHPS genome walking was attempted and DHPS and DHFR
primer annealing sites explored. Amplified DHPS and DHFR genes were cloned and
sequenced.
Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtime
PCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review of
clinical records suggested a high rate of false-positive IF results. P. jirovecii was
detected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositive
HIV-negative patients. P. jirovecii was detected in 45% HIV-negative patients. The most prevalent ITS type was Eg. Four new combinations: Eo, Je, Ge,
No; 11 new ITS1 and 13 new ITS2 sequences were identified. A new ITS2 type was
detected in three patients and designated u. More than one strain type was detected
in 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2
nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currently
employed primers amplified DHPS and DHFR genes from 53 and 27 specimens,
respectively. Newly designed DHPS primers increased detection in 3 / 28 previously
DHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified and
sequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFR
Ala67Val was detected in three specimens and a new DHFR genotype (Arg59Gly;
C278T) was demonstrated.
Conclusions: The study emphasises the need to evaluate PCR primers against local
strains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF and
discordant results resolved with clinical evaluation. Co-infection with P. jirovecii and
M. tuberculosis occurs in South Africa, and treatment for both pathogens is
recommended when demonstrated by the laboratory. ITS genotyping employing
statistical parsimony network analysis suggests type Eg as major ancestral
haplotype, and supports recombination contributing to strain diversity worldwide.
DHPS mutations may signal emergence of resistance to cotrimoxazole in South
Africa, however, low sensitivity of primers limits surveillance efforts.
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Enhancing analytical capability of piezoelectric quartz crystal and capillary electrophoresis in environmental analysis using polymerasechain reaction, molecularly imprinted polymers and nanotechnologySun, Hui, 孫慧 January 2006 (has links)
published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy
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Methods for serological and PCR detection of Salmonella enteritidis in chickens.Meyer, Brendan. 08 November 2013 (has links)
Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently
one of the leading causes of human food poisoning in the world. It is believed that
contaminated poultry products, especially eggs and egg products, have been responsible
for the dramatic increase in the incidence of this Salmonella serotype. Detection of
S. entertidis has conventionally involved bacteriological examination of samples, yet these
procedures are time-consuming which could lead to the rapid spread of S. enteritidis
through commercial flocks and potentially cause a human health risk. A number of
alternative detection techniques, mostly based on serological methods, have been reported
as effective diagnostic assays. However, some of these reports have not been supported by
representations of SDS-PAGE gels or Western blots. The objective of this study was the
evaluation of these serological techniques as well as a PCR amplification technique, which
has been reported to show promising results as a diagnostic method. The techniques
discussed in these reports were evaluated with regards to how rapid they were, their
specificity and their potential for use in local diagnostic laboratories.
Antigens from the outer surface of S. enteritidis were purified by several methods and their
antigenicity was tested by separating the antigens by means of SDS-PAGE, followed by
Western blotting using sera of chickens infected with S. enteritidis. A high degree of cross
reactivity was observed with many of the antigens tested, especially the
lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) which had previously
been reported as containing antigens which could be used for specific detection of
S. enteritidis. This cross-reactivity could be explained by the conserved nature of many of
the LPS and OMP antigens among the Salmonella serotypes tested. A fimbrial antigen,
SEF14, which has been reported as a novel antigen, was seen as a prominent band at 14.3
kDa and was found to react with antibodies against S. enteritidis, yet not to the specificity
levels described in previous reports.
PCR amplification of the sefA gene sequence, which encodes for the SEF14 fimbrial
antigen, was found to give a predicted product of 310 bp when using a previously
described oligonucleotide primer pair. This amplified product was found to be specific for
S. enteritidis and other serogroup D Salmonella serotypes that are not poultry pathogens The cross-reactivity observed with many of the serological techniques used in this study,
meant that detection of S. enteritidis infection in chickens was considerably hindered.
However, the identification of further novel antigens by serological means, could result in the development of new vaccines. The specificity and speed afforded by PCR amplification indicated that this technique showed excellent potential for use in local diagnostic laboratories. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.
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Detection of NheA from Bacillus spp. in food and soil isolates using real-time and rep-PCR / Detection of non-hemolytic enterotoxin A from Bacillus spp. in food and soil isolates using real-time and rep-polymerase chain reactionBeer, Matthew R. 06 August 2011 (has links)
Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology
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Exploration, quantification, and mitigation of systematic error in high-throughput approaches to gene-expression profiling : implications for data reproducibilityKitchen, Robert Raymond January 2011 (has links)
Technological and methodological advances in the fields of medical and life-sciences have, over the last 25 years, revolutionised the way in which cellular activity is measured at the molecular level. Three such advances have provided a means of accurately and rapidly quantifying mRNA, from the development of quantitative Polymerase Chain Reaction (qPCR), to DNA microarrays, and second-generation RNA-sequencing (RNA-seq). Despite consistent improvements in measurement precision and sample throughput, the data generated continue to be a ffected by high levels of variability due to the use of biologically distinct experimental subjects, practical restrictions necessitating the use of small sample sizes, and technical noise introduced during frequently complex sample preparation and analysis procedures. A series of experiments were performed during this project to pro le sources of technical noise in each of these three techniques, with the aim of using the information to produce more accurate and more reliable results. The mechanisms for the introduction of confounding noise in these experiments are highly unpredictable. The variance structure of a qPCR experiment, for example, depends on the particular tissue-type and gene under assessment while expression data obtained by microarray can be greatly influenced by the day on which each array was processed and scanned. RNA-seq, on the other hand, produces data that appear very consistent in terms of differences between technical replicates, however there exist large differences when results are compared against those reported by microarray, which require careful interpretation. It is demonstrated in this thesis that by quantifying some of the major sources of noise in an experiment and utilising compensation mechanisms, either pre- or post-hoc, researchers are better equipped to perform experiments that are more robust, more accurate, and more consistent.
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Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood SamplesGomes, Cláudia, Martinez Puchol, Sandra, Pons, Maria J., Bazán, Jorge, Tinco, Carmen, Del Valle Mendoza, Juana Mercedes, Ruiz, Joaquim 09 March 2016 (has links)
Background
The lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches.
Methodology/Principal Findings
We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay.
Methodology/Principal
Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay.
Conclusions/Significance
From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.
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