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1
Desvars, Amélie. "Épidémiologie d'une zoonose, la leptospirose, dans deux îles de l'océan Indien, la Réunion et Mayotte - Étude comparée du rôle de différentes espèces sauvages et domestiques." Electronic Thesis or Diss., La Réunion, 2012. http://www.theses.fr/2012LARE0039.
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La leptospirose est une zoonose de répartition mondiale dont les formes graves peuvent être mortelles pour l'Homme. Tous les mammifères sont susceptibles d'être réservoirs, la connaissance des hôtes réservoirs de Leptospira est essentielle à la mise en place de mesures de prévention. L'objectif de ce travail était de conduire une étude épidémiologique descriptive transversale de la leptospirose animale dans deux îles de l'Océan Indien : La Réunion et Mayotte. À La Réunion, 579 animaux appartenant à 13 espèces ont été prélevés. Nous montrons que la maladie circule chez l'ensemble des espèces, sa séroprévalence varie de 15,7% chez les tangues à 79,8% chez les rats, tandis que la prévalence du portage rénal varie de 0% chez les tangues à 84,6% chez les souris. Ce travail est le premier qui quantifie la charge bactérienne rénale d'animaux infectés naturellement. À Mayotte, 292 animaux ont été étudiés. La séroprévalence est de 2% chez les lémuriens, 10,2% chez les roussettes, 11,2% chez les rats noirs alors qu'elle est supérieure à 85% chez les chiens. Nous confirmons l'absence du sérogroupe Icterohaemorrhagiae à Mayotte et montrons que le sérogroupe Mini est le principal sérogroupe circulant chez les rats et les chiens non vaccinés. La prévalence du portage rénal a été estimée à 29,8% chez les rats. Les résultats du séquençage montrent une grande diversité génétique des souches circulant chez le rat ainsi qu'une parfaite homologie avec celles isolées chez des patients mahorais, désignant le rat noir comme source majeure de contamination pour l'Homme. Dans des zones tropicales comme La Réunion et Mayotte, la prophylaxie doit considérer l'écosystème dans sa globalitéLeptospirosis is a widespread zoonosis which could be letal for humans. All mammals could be reservoir of the bacteria in their kidneys and knowledge about the maintenance hosts is essential to improve preventive measures. The aim of this work was to conduct an epidemiological descriptive transversal study on animal leptospirosis on two Indian Ocean islands: Reunion and Mayotte. In Reunion Island, we studied 579 mammals belonging to 13 species. Results showedthat seroprevalence of leptospirosis varied greatly regarding the species, from 15.7% in the insectivorous tenrecs, to 79.5% in rats and prevalence of the renal carriage varied from 0% in tenrecs to 84.6% in mice. This is the first report that evaluates the concentration of leptospires in the kidney tissue of naturally infected mammals. In Mayotte, 292 animals were studied. We showed that the seroprevalence was 2% in lemurs, 10.2% in flying foxes, 11.2% in black rats, while it was over 85% in domestic and stray dogs. We showed that Mini was the most prevalent serogroup found in rats and nonvaccinated dogs and corroborated recent findings showing that serogroup Icterohaemorrhagiae was not present in Mayotte. We reported 29.8% of renal carriage amongst rats. DNA sequencing showed a great diversity among Leptospira strains circulating within rat population and a perfect homology with the strains isolated from ill patients in Mayotte. These results strongly suggest that black rats are the main source of human contamination in Mayotte. These data have practical applications in human and veterinary medicine. In tropical areas such as Reunion and Mayotte islands, prophylaxia should be considered at the ecosystem level
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Zhang, Yan. "Frequent RASSF1A gene promoter hypermethylation in breast cancer." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-63611.
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Andalo, Alice. "Analisi quantitativa dell'espressione genica mediante real-time rt-pcr." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amslaurea.unibo.it/8450/.
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Il primo capitolo di questo lavoro di tesi introduce i concetti di biologia necessari per comprendere il fenomeno dell’espressione genica. Il secondo capitolo descrive i metodi e le tecniche di laboratorio utilizzate per ottenere il cDNA, il materiale genetico che verrà amplificato nella real-time PCR. Nel terzo capitolo si descrive la tecnica di real-time PCR, partendo da una descrizione della PCR convenzionale fino a delineare le caratteristiche della sua evoluzione in real-time PCR. Si prosegue con la spiegazione del principio fisico alla base della tecnica e delle molecole necessarie (fluorofori e sonde) per realizzarla; infine si descrive l’hardware e il software dello strumento. Il quarto capitolo presenta le tecniche di analisi del segnale che utilizzano metodi di quantificazione assoluta o relativa. Infine nel quinto capitolo è presentato un caso di studio, cioè un’analisi di espressione genica con real-time PCR condotta durante l’esperienza di tirocinio presso il laboratorio ICM. e delle molecole necessarie (fluorofori e sonde) per realizzarla; infine si descrive l’hardware e il software dello strumento. Il quarto capitolo presenta le tecniche di analisi del segnale che utilizzano metodi di quantificazione assoluta o relativa. Infine nel quinto capitolo è presentato un caso di studio, cioè un’analisi di espressione genica con real-time PCR condotta durante l’esperienza di tirocinio presso il laboratorio ICM.
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Tichopád, Aleš. "Quantitative real-time RT-PCR based transcriptomics improvement of evaluation methods /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972765069.
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Steyn, HC, A. Pretorius, and CME McCrindle. "A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20." Elsevier, 2008. http://encore.tut.ac.za/iii/cpro/DigitalItemViewPage.external?sp=1000379.
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Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by
Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater
take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCRTaqMan probe assay to
detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E.
ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A.,
Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E.,
Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A.,
2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy
number. PNAS 102, 838–843]. The pCS20 quantitative real-time PCRTaqMan probe was compared to the currently used pCS20
PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood
from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCRTaqMan probe was the
most sensitive assay detecting seven copies of DNA/ml of cell culture. All three assays, however, cross react with Ehrlichia canis
and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and
pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile
reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus,
because this new quantitative pCS20 real-time PCRTaqMan probe assay was the most sensitive and can be performed within 2 h
it is an effective assay for epidemiological surveillance and monitoring of infected animals.
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Leopold, Luciana Eleanor Dittmer Dirk Peter. "Development of real-time PCR assays for the quantitative detection of herpesviruses." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1487.
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Thesis (M.S.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Master of Science in the Curriculum of Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.
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Jungebloud, Anke. "Untersuchung der Genexpression in Aspergillus niger mittels Echtzeit-PCR." Paderborn FIT-Verl. für Innovation und Technologietransfer, 2007. http://www.gbv.de/dms/bs/toc/533996201.pdf.
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Rosa, Stefanie Ulrike. ""Real-Time-PCR-Untersuchungen zur Persistenz von infektiösen Toxoplasma-gondii-Dauerstadien in Rohwurst-Erzeugnissen"." Giessen VVB Laufersweiler, 2009. http://d-nb.info/996004408/04.
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Lorenz, Andreas. "Quantitative Real-time PCR zum spezifischen Nachweis transrenaler DNA des Mycobacterium tuberculosis complex." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-115143.
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Utokaparch, Soraya. "Development of a standardized quantitative real time PCR panel for respiratory viral diagnosis." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/31339.
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Traditional viral diagnostics such as viral culture and various serological techniques tend
to be slow, insensitive and labour-intensive. A large proportion of viral pathogens still go
undetected using these techniques. This thesis concerns the development of a rapid and
sensitive technique - standardized real-time quantitative PCR. Individual qPCR assays
and synthetic plasmid controls were developed for 12 common respiratory viruses
including influenza types A and B, parainfluenza (PIV)-l, -2 and -3, respiratory syncytial
virus (RSV) A and B, metapneumovirus (MPV), human coronavirus (HCoV) 229E and
OC43, human rhinovirus (HRV) and adenovirus. A reference gene assay using
hypoxanthine phosphoribosyl transferase (HPRT) was also developed. A retrospective
analysis on nasopharyngeal aspirates from patients previously diagnosed was conducted.
The results demonstrated that the respiratory viral qPCR panel was sensitive, efficient,
and had a large dynamic range of detection. Some cross-reactivity was noted for HRV
with an enterovirus (coxsackievirus B3). HPRT proved to be a stable reference gene with
the additional benefit that qPCR viral loads could be interpreted based on copy number
per unit volume of specimen. One hundred culture negative specimens were examined
and viral nucleic acid was amplified in 43 of them. There was a statistically significant
relationship between viral load and whether or not the same specimen was positive by
culture for influenza A , PIV-3, RSV A and B, HRV and adenovirus. Mean viral load was
highest in patients with lower respiratory tract infections (LRTI) compared to those with
fever or upper respiratory tract infections (URTI) and 95% confidence interval (CI)
between these patients did not overlap. These results suggest that patients with more
severe clinical disease had higher viral loads. This study highlights the developmental
phase of a technique that has the potential to increase the detection rate of viral pathogens
involved in respiratory illnesses.Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate
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Kessler, Yvonne. "Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues /." [S.l.] : [s.n.], 2009. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000292626.
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Sieuwerts, Anita Maria. "Prognostic and predictive testing of molecular markers in breast by real-time quantitative PCR." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2007. http://hdl.handle.net/1765/10736.
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張綺雲 and Yee-wan Cheung. "Quantitative analysis of hTERT mRNA expression in gestational trophoblastic disease by real-time PCR." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31970436.
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Cheung, Yee-wan. "Quantitative analysis of hTERT mRNA expression in gestational trophoblastic disease by real-time PCR." Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25139198.
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Grimm, Julia. "Untersuchungen zur Prävalenz von Mycoplasma suis in Deutschland sowie vergleichende Untersuchungen zwischen real-time Polymerasekettenreaktion und Akridin-Ausstrich bezüglich ihrer Sensitivität und Spezifität." München Verl. Dr. Hut, 2008. http://d-nb.info/988229315/04.
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Nascimento, Cristiane Santos. "Uso de método de biologia molecular quantitativo (PCR real-time) na avaliação da carga parasitária em cães naturalmente infectados por Leishmania sp." s. n, 2011. https://www.arca.fiocruz.br/handle/icict/4152.
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Cristiane Santos Nascimento Uso de método de biologia molecular....pdf: 1323686 bytes, checksum: caf3acf13d85858d02cc05e45a821e9c (MD5)Made available in DSpace on 2012-06-25T21:03:51Z (GMT). No. of bitstreams: 1 Cristiane Santos Nascimento Uso de método de biologia molecular....pdf: 1323686 bytes, checksum: caf3acf13d85858d02cc05e45a821e9c (MD5) Previous issue date: 2011
Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
INTRODUÇÃO: A leishmaniose visceral humana (LVH) é uma importante causa de morbidade e mortalidade no Brasil. Apesar dos avanços no conhecimento da epidemiologia da LVH, ainda existem lacunas importantes nas informações sobre os principais reservatórios desta zoonose. A técnica validada para avaliação da infectividade de reservatórios, xenodiagnóstico, é um método laborioso, demorado e difícil de executar, portanto, inapropriado para a triagem de grande número de animais. A padronização de método capaz de quantificar a carga parasitária presente em diferentes tecidos pode oferecer respostas importantes sobre a epidemiologia e a prevenção da LVH. OBJETIVO: Avaliar a carga parasitária em diferentes amostras biológicas de cães naturalmente infectados por Leishmania chagasi, utilizando método de biologia molecular quantitativo, PCR real-time (qPCR). MÉTODOS:Entre nov/2004 e abr/2007, foram realizados seis inquéritos soro-epidemiológicos em duas áreas endêmicas para LVH. Os cães soropositivos foram eutanasiados e submetidos a: exame de cultura, parasitológico direto e exame histológico para confirmação da infecção. Amostras de sangue periférico e fragmento de pele foram coletadas em todos os animais para determinação da carga parasitária. Adicionalmente, coletou-se também swab da conjuntiva, aspirado de medula óssea e linfonodo para realização do teste de qPCR nos cães incluídos no último inquérito (abr/2007). A técnica de qPCR foi padronizada utilizando um par de primers LEIF e LEIR e sonda LEIP selecionados no gene SSu rRNA. A seleção dos primers e sonda foi realizada utilizando o programa Primer Express (Perkin-Elmer-Applied Biosystems). A sonda fluorogênica foi sintetizada utilizando uma molécula FAM ligada na extremidade 5‟ e TAMRA ligada à extremidade 3‟(Perkin-Elmer -Applied Biosystems). Para determinar a carga parasitária foi realizada curva padrão com o DNA obtido da cultura de L. chagasi em concentrações variando de 101 a 107 parasitas/ml. Cada ponto da curva foi testado em triplicata. RESULTADOS: Dos 98 cães soropositivos identificados, foi detectado DNA de Leishmania em 57% das amostras de sangue total, em 56% das amostras de pele e em 100% das amostras de medula óssea, linfonodos e swab da conjuntiva. A carga parasitária em sangue periférico e swab da conjuntiva não ultrapassou 103 parasitas/ml sendo mais comumente detectado 1 a 10 parasitas/ml. Por outro lado, em pele, medula óssea e linfonodos a carga parasitária passou de 104 parasitas/ml, além disso, as quantidades de DNA detectadas se distribuíram com maior freqüência na categoria acima de 104 parasitas/ml, notadamente em amostras de linfonodos. CONCLUSÕES: O qPCR apresentou alta sensibilidade nas amostras biológicas estudadas, particularmente em linfonodos , medula óssea e pele. Nossos resultados indicam que o qPCR pode ser utilizado numa variedade de amostras biológicas para a quantificação da carga parasitária de cães naturalmente infectados por Leishmania sp. Estudos de validação do qPCR para avaliar a capacidade de reservatórios da LV, em lugar do xenodiagnóstico, e para investigar o papel do qPCR na triagem de cães em programas de controle/prevenção da LV devem ser conduzidos.
INTRODUCTION: Human visceral leishmaniasis (LVH) is an important cause of morbidity and mortality in Brazil. Despite advances in knowledge of the epidemiology of LVH, there are still important gaps in information on the main reservoirs of this zoonotic disease. The validated technique for assessing the infectivity of reservoirs, xenodiagnosis, is laborious, time consuming and difficult to implement, therefore, inappropriate for screening large numbers of animals. A standardized method to quantify the parasite load present in different tissues may provide important answers on the epidemiology and prevention of LVH. OBJECTIVE: To assess the parasite load in different biological samples from dogs naturally infected by Leishmania chagasi using a molecular biology quantitative method, real-time PCR (qPCR). METHODS: From nov/2004 to apr/2007, six seroepidemiological surveys were conducted in two endemic areas for LVH. The seropositive dogs were euthanized and submitted to: culture, direct parasitological and histological examination to confirm infection. Blood samples and skin fragments were collected in all animals to determine the parasite load. Additionally, conjuntival swabs, bone marrow and lymph node aspirates were also collected to do qPCR in dogs included in the last survey (apr/2007). The qPCR technique was standardized using a pair of primers and probe and LEIF /LEIR and LEIP selected in the SSU rRNA gene. The selection of primers and probe was performed using the program Primer Express (Perkin-Elmer-Applied Biosystems). The fluorogenic probe was synthesized using a FAM molecule attached at the 5 'end and TAMRA linked to the 3' end (Perkin-Elmer-Applied Biosystems). In order to determine the parasite load a DNA standard curve was plotted with DNA obtained from L. chagasi culture in concentrations ranging from 101 to 107 parasites/ ml. Each point on the curve was tested in triplicate. RESULTS: Of the 98 seropositive dogs identified Leishmania DNA was detected in 57% of the whole blood samples, 56% of the skin samples and 100% of bone marrow, lymph nodes and conjuntival swabs samples. The parasite load in peripheral blood and conjuntival swab did not exceed 103 parasites/ml, and was more commonly in the range of 1-10 parasites /ml. On the other hand, skin, bone marrow and lymphnode parasite burden exceeded 104 parasites / ml, in addition, the quantities of DNA detected were distributed more frequently in the category above 104 parasites/ml, especially in lymphnodes samples. CONCLUSIONS: qPCR showed high sensitivity in biological samples studied, particularly in lymphnodes, bone marrow and skin. Our results indicate that qPCR could be used in a variety of biological samples to quantify the parasite load in dogs naturally infected by Leishmania sp. qPCR validation studies to assess potential reservoirs for VL (replacing xenodiagnosis), and to investigate the role of qPCR in dog screening programs for the control/prevention of LV should be conducted.
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Handy, Sara Marie. "Investigations of the ecology of Delaware Inland Bay harmful algae utilizing quantitative real-time PCR." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 152 p, 2007. http://proquest.umi.com/pqdweb?did=1362527691&sid=13&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Noll, Lance. "Escherichia coli O157: detection and quantification in cattle feces by quantitative PCR, conventional PCR, and culture methods." Thesis, Kansas State University, 2015. http://hdl.handle.net/2097/18923.
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Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
T. G. Nagaraja
Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.
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Richterich, Peter. "Expression des Insulin-like-growth-factor-Systems in Rinderplazentomen im Verlauf der normalen Gravidität sowie bei Graviditäten aus In-vitro-Embryoproduktion mit dem pathologischen Erscheinungsbild "Large Offspring Syndrome"." Giessen VVB Laufersweiler, 2008. http://d-nb.info/993478840/04.
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Kemmerling, Kirsten. "Chlamydieninfektionen bei Milchkühen in Nordrhein-Westfalen, Prävalenz, Risikofaktoren, Kennziffern und Vorhersagewahrscheinlichkeiten." Göttingen Cuvillier, 2009. http://d-nb.info/1000908437/04.
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Miller, Carin R. "Developmental Gene Expression in the Small Intestine of Chickens from Lines Divergently Selected for High or Low Juvenile Body Weight." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/35168.
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Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation and therefore the expression of these transporters can influence the overall nutrient status as well as the growth and development of the animal. This thesis examined correlated responses to selection in the developmental gene expression of the peptide transporter PepT1, the glutamate/aspartate transporter EAAT3, the sodium-dependent glucose transporter SGLT1, and the fructose transporter GLUT5 in the small intestine of chickens from lines divergently selected for high (HH) or low (LL) eight-week body weight and their reciprocal crosses, (HL and LH). Chicks were weighed and killed on embryonic day 20 (E20), day of hatch (DOH with no access to feed), and days 3 (D3), 7(D7), and 14 (D14) post hatch. Duodenum, jejunum, ileum and liver were collected. DNA extracted from liver was used to sex birds by PCR. RNA was extracted from the intestinal segments of four males and four females from each mating combination (MC) and time point except E20 HL males (n = 3) and D7 LL females (n = 2). Expression of nutrient transporters was assayed by real-time PCR using the relative quantification method. In comparing HH and LL males and females there was a line by segment interaction in PepT1 gene expression, with no segment difference in HH and greatest expression in the ileum of the LL (P < 0.05). There was also a MC by age by sex interaction for PepT1 gene expression (P < 0.0001) with peak gene expression occurring on DOH for LL females, on D7 for HH females, on D7 for LL males and D14 for HH males. Overall, females had greater EAAT3 expression (P < 0.03). Gene expression of EAAT3 was greatest in the ileum, intermediate in the jejunum, and least in the duodenum (P < 0.0007). There was an age by segment interaction for EAAT3 expression (P = 0.0002) and a MC by segment interaction (P < 0.02), with LL having greater expression than HH in the ileum. Females had greater SGLT1 expression than males (P < 0.0001). There was a sex by age interaction for the expression of SGLT1 (P < 0.0001). Females induced SGLT1 expression on DOH and maintained this level through D14, while males gradually increased expression through D7 and decreased expression by D14. These results indicate that expression of PepT1, EAAT3, SGLT1 are differentially expressed in male and female chickens regardless of selection for high or low juvenile body weight. These results also show a sexual dimorphism in the capacity to absorb peptides, anionic amino acids, and glucose from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. In comparing male HH, HL, LH, and LL chicks, overall LL had the greatest level of expression (P <0.06), HH had the least level of expression (P < 0.006) and HL and LH had intermediate levels of expression (P < 0.06). Greatest PepT1 gene was expression in the ileum (P < 0.0003) and there was a MC by segment interaction with expression increasing from duodenum to ileum in LL, but there was no segment difference in any other MC (P < 0.08). Within each intestinal segment there was a MC difference (P < 0.02). There was an effect of sire for PepT1 expression, with progeny from low weight selected sires (LWS) having greater expression than progeny from high weight selected (HWS) sires (P = 0.0008). There was no difference between intestinal segments in progeny from HWS sires, however, greatest PepT1 gene expression was seen in the ileum of progeny from LWS sires (P < 0.0001). Overall, expression of EAAT3 was greatest in the ileum, intermediate in the jejunum and least in the ileum (P < 0.0001) and there was a segment by age interaction for EAAT3 expression (P < 0.0001). In all MCs except HH, EAAT3 gene expression increased from duodenum to ileum (P < 0.08). Within the ileum, the LL had greatest EAAT3 gene expression, LH and HL had intermediate gene expression, and HH had least expression (P < 0.08). Expression of SGLT1 gradually increased through D7 and decreased by D14 (P < 0.0001) and overall, was greatest in the distal small intestine (P < 0.0001). There was a MC by segment interaction, with SGLT1 gene expression being greatest in the distal small intestine in LL, LH, and HL, but greatest in the jejunum of HH (P < 0.04). Within the ileum, LL had greater SGLT1 gene expression than HH (P < 0.06). Overall, greatest GLUT5 expression was in the distal small intestine (P < 0.0001) and there was a MC by segment interaction, with expression being greatest in the distal small intestine in LL and HL (P < 0.02), greatest in the ileum of LH (P < 0.08), and greatest in the jejunum of HH (P < 0.09). Within the ileum there was a MC difference (P < 0.07). These results indicate that selection for high or low juvenile body weight may have influenced the gene expression pattern of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens.Master of Science
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Vatanparast, Raha [Verfasser]. "Entwicklung einer quantitativen Real-Time PCR Methode zur Bestimmung der Gesamtkeimzahl in Geflügelfleisch / Raha Vatanparast." Hannover : Technische Informationsbibliothek (TIB), 2016. http://d-nb.info/1119907004/34.
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Herbel, Stefan [Verfasser]. "Using GroEL (hsp60) for a Specific Quantification of Lactobacilli by Quantitative Real-Time PCR / Stefan Herbel." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/107826161X/34.
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Conway, Crystal A. "Study of Secondary Metabolite Gene Expression in Marine Microbial Co-Cultures Using Quantitative Real-Time PCR." NSUWorks, 2010. http://nsuworks.nova.edu/occ_stuetd/222.
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Interactions among microbial organisms often cannot be observed directly, but they can be inferred genetically using new molecular techniques. The analysis of secondary metabolite gene expression produced by co-cultured marine microbial species allows us to see how these organisms interact with one another when kept in the same environment. Co-cultures of three different strains of marine bacteria, P. aeruginosa PAO1, Roseobacter denitrificans OCH114, and Salinispora arenicola CNS-205 were grown in a laboratory setting, and using the Real-Time qPCR method gene expression levels of two different secondary metabolite producing genes from each organism was accessed across three time points. P. aeruginosa PAO1’s secondary metabolite genes RdhA and PhzH stayed repressed through all co-cultures and time points in this study, and Roseobacter denitrificans OCH-114’s secondary metabolite genes metallo-beta-lactamase and DMSP lyase were up-regulated after the 30 minute time point in the P. aeruginosa-R. denitrificans co-culture and at the 0 minute time point in the R. denitrificans-S. arenicola co-culture.
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White, James David dvm. "Real-Time Quantitative PCR of tet (C), in 2 Swine Populations: Antibiotic Free versus Conventionally Reared." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437046111.
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Herbel, Stefan Roland [Verfasser]. "Using GroEL (hsp60) for a Specific Quantification of Lactobacilli by Quantitative Real-Time PCR / Stefan Herbel." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/107826161X/34.
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Chagas, Júnior Adenizar Delgado das. "Avanços no conhecimento da imunopatogênese da leptospirose e a aplicação do método do imprint como ferramenta qualitativa e quantitativa de leptospiras." Centro de Pesquisas Gonçalo Moniz, 2014. https://www.arca.fiocruz.br/handle/icict/7628.
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Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil
A leptospirose é uma zoonose causada por espiroquetas patogênicas pertencentes ao gênero Leptospira. O modelo da doença em camundongos tem vantagens devido à ampla gama de ferramentas genéticas e imunológicas disponíveis para pesquisas básicas. A maior limitação na conduta clínica e na pesquisa experimental da leptospirose é o fraco desempenho dos métodos disponíveis para detecção direta e para quantificação de leptospiras. Foi incluído nesta tese um conjunto de três manuscritos que visam investigar o desfecho da infecção pela cepa virulenta de Leptospira interrogans nas linhagens de camundongos selvagens (A, CBA, BALB/c e C57BL/6), em camundongos óxido nítrico sintase induzível (iNOS) Knockout (KO), camundongos gene ativador de recombinação 1 (RAG1) KO , camundongos CB17 com imunodeficiência combinada grave (SCID), e os seus respectivos controles selvagens C57BL/6 e BALB/c. Investigar a confiabilidade do método de quantificação do imprint (IM), comparando os resultados obtidos com esta técnica aos obtidos com a utilização do PCR em tempo real (qPCR) para detectar e quantificar leptospiras em amostras de rim de ratos e hamsters experimentalmente infectados. Como esperado, nenhuma das linhagens de camundongos selvagens foram suscetíveis à leptospirose letal. A linhagem A e C57BL/6 exibiram altas cargas de Leptospira nas amostras de rim e as linhagens CBA e C57BL/6 desenvolveram lesões inflamatórias graves, enquanto a linhagem BALB/c provou ser a mais resistente apresentando leptospirose subclínica. Os camundongos iNOS KO e selvagem sobreviveram sem sintomas clínicos de leptospirose. A frequência e gravidade das nefrites foram significantemente menores nos camundongos iNOS KO. Todos os animais RAG1 KO e SCID morreram de leptospirose aguda, enquanto que todos os camundongos selvagens sobreviveram. A hemorragia pulmonar foi observada em 57 e 94% dos camundongos RAG 1 KO e em 83 e 100% dos camundongos SCID, usando doses de inóculos de 107 e 106 leptospiras, respectivamente. Não houve evidências de hemorragia pulmonar nos controles selvagens. Nos modelos de infecção agudo e crônico, houve correlação positiva estatisticamente significante (P < 0,05) na quantificação de leptospiras pelos métodos do qPCR e do IM. Como conclusão geral, a linhagem de camundongos A pode ser a linhagem de escolha em estudos na qual se pretende recuperar um grande número de leptospiras de rins colonizados. As linhagens CBA e C57BL/6 desenvolveram, com maior frequência, lesões inflamatórias e podem ser as mais adequadas para estudos de leptospirose associados com nefrite intersticial. A linhagem BALB/c é a mais indicada para estudar mecanismos que envolvam a imunidade inata e/ou a rápida resposta imune adaptativa. A ausência do gene do iNOS no modelo murino resultou em uma diminuição significativa da suscetibilidade para o desenvolvimento da nefrite intersticial. Além disso, a ausência de linfócitos B e T funcionais não impediu a ocorrência de hemorragia pulmonar. Estes dados fornecem fortes evidências de que a hemorragia pulmonar na leptospirose não está relacionada apenas a mecanismos autoimunes. Para a detecção e quantificação de leptospiras o método do imprint foi equivalente ao qPCR.
Leptospirosis is a zoonosis caused by pathogenic spirochaetes belonging to the genus Leptospira. The mouse disease model is advantagous due to the broad array of immunological and genetic tools available for basic research. A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods in the direct detection and quantification of leptospires. This thesis includes three manuscripts that investigate the outcome of infection by a virulent strain of Leptospira interrogans in wildtype mice strains: A, CBA, BALB/c and C57BL/6; in iNOS knockout (KO) mice, recombination activating gene 1 (RAG1) KO mice and CB17 severe combined immunodeficiency (SCID) mice. To investigate whether the imprint method (IM) of quantification was reliable we compared it with against real time PCR (qPCR) for the detection and quantification of leptospires in kidney samples from rats and hamsters. As expected, none of the wildtype mice were susceptible to lethal leptospirosis. The A and C57BL/6 strains exhibited high leptospiral loads in the kidney samples and the CBA and C57BL/6 strains developed severe inflammatory lesions, whilst the BALB/c strain proved to be the most resistant to subclinical leptospirosis. The iNOS KO mice survived with no clinical symptoms of leptospirosis. The frequency and severity of nephritis was significantly lower in the iNOS KO mice. All of the RAG 1 KO and SCID animals died of acute leptospirosis, whereas all of the wildtype mice survived. Pulmonary haemorrhage was observed in 57 and 94% of Rag1 KO mice and in 83 and 100% of SCID mice, using inoculum doses of 107 and 106 leptospires, respectively. There was no evidence of pulmonary haemorrhage in the wildtype controls. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P < 0.05). In conclusion, the mouse A strain would be the strain of choice in studies requiring the recovery of a large number of leptospires from colonized kidneys. The CBA and C57BL/6 strains developed more inflammatory lesions and would be the most suitable for studies of leptospirosis associated with interstitial nephritis. The BALB/c strain appeared to be the most suitable for studying mechanisms involving innate immunity and/or rapid adaptive immune response. The absence of the iNOS gene in the murine model resulted in a significant decrease in susceptibility to the development of interstitial nephritis. Furthermore, the absence of functional B and T lymphocytes did not prevent the occurrence of pulmonary hemorrhage. These data provide strong evidence that pulmonary hemorrhage in leptospirosis is not only related to autoimmune mechanisms. For the detection and quantification of leptospires the imprint method was quivalent to that of qPCR. Keywords:
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Wehrheim, Kerstin [Verfasser]. "Möglichkeiten und Grenzen der klonspezifischen, quantitativen Real-Time-PCR bei Patienten mit Sézary Syndrom / Kerstin Wehrheim." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2014. http://d-nb.info/1051812356/34.
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Pinto, Maria Joana Amado Teixeira. "An integrated BAC approach to the genomics of cork formation." Master's thesis, ISA, 2013. http://hdl.handle.net/10400.5/6107.
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Mestrado em Biologia Funcional - Instituto Superior de AgronomiaCork is produced by Quercus suber, an evergreen oak with major economic and environmental importance in Portugal. Suberin is the main component of cork but little is known about the molecular processes underlying suberin biosynthesis. We screened a Quercus suber bacterial artificial chromosome (BAC) library for the presence of relevant genes for suberin biosynthesis through high-density BAC filters hybridizations and we validated hybridization positive clones by Colony PCR. We analyzed BAC-end sequences (BESs) to characterize the structure and composition of the cork oak genome. And we hybridized BAC DNA with mitotic cork oak chromosomes to obtain a cytogenetic map of Q. suber. Finally, we analyzed expression profiles of candidate genes of cork samples from cork oaks with different cork qualities, using quantitative real-time PCR. We identified several genes involved in suberin biosynthesis pathways. BESs analysis showed high identity with coding regions and suggest the existence of conserved regions within the Fagacea family. We found strong evidence that the genes Cyp 86A1 and GDSL involved in lipid suberin biosynthesis pathway are closely located in the genome. However, we did not succeed in mapping genes of interest in the cork oak genome and we did not find any relation between the genes relative expression and cork quality
The work was partially supported by a grant from the Fundação para a Ciência e Tecnologia (FCT), project SuberGene (PTDC/AGR-GPL/101785/2008, Genomics of Cork Formation: an integrated approach of cork quality
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Lambers, Christopher. "Nachweis von K-ras Mutationen in kolorektalen Tumoren und regionalen Lymphknoten durch allelspezifische, quantitative real-time PCR." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=972321713.
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Chan, Ho-yan Mandy, and 陳可欣. "Development of multiplex quantitative real-time PCR for detection of common viral infections of central nervous system." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46631859.
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Reinbold, James Brandon. "Application of real-time quantitative RT-PCR for improving the diagnosis, treatment, and control of bovine Anaplasmosis." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/2305.
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Silva, Thaís Galhardo Egreja Ribeiro da. "Raquitismo da soqueira de cana-de-açúcar: transmissão do patógeno via sementes e avaliação quantitativa de seu controle através da termoterapia de toletes." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-17122013-112133/.
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Devido à sua múltipla utilidade, a cana-de-açúcar (Saccharum spp.) é amplamente cultivada em mais de 127 países tropicais e subtropicais. No Brasil, a cultura apresentou expansão em área plantada nos últimos anos em virtude da crescente demanda por biocombustíveis. Porém, a produção de mudas sadias tende a ser um fator limitante a este aumento de demanda, já que diversas doenças são transmitidas por toletes contaminados. Dentre estas, destaca-se o raquitismo da soqueira (RSD), causado pela bactéria fastidiosa Leifsonia xyli subsp. xyli (Lxx) (KAO e DAMANN, 1980). De acordo com a literatura, a transmissão de Lxx se dá através de toletes contaminados e/ou instrumentos de corte, não havendo relatos sobre sua transmissão por sementes. Desta forma, seu controle se baseia no princípio da exclusão, através do uso de toletes tratados termicamente como fonte de material propagativo. Porém, não se conhece o efeito do tratamento no título de Lxx, uma vez que os relatos de eficiência da termoterapia baseiam-se em avaliações qualitativas. Em face destas informações, este estudo objetivou avaliar a transmissão de Leifsonia xyli subsp. xyli por sementes e também quantificar o efeito da termoterapia no título de Lxx em duas variedades de cana-de-açúcar através da PCR quantitativa em tempo real. No ensaio de transmissão via sementes, a bactéria foi detectada em altas incidências (>87%) em plantas de 90 dias oriundas de sementes coletadas de plantas infectadas e em quantidades estimadas superiores a 51 células por 100ng de DNA total de sementes. Além disso, aos 270 dias após o plantio, a bactéria foi isolada de 6 plantas. A identidade das colônias foi comprovada através de PCR convencional utilizando primers específicos para Lxx. Dois ensaios foram conduzidos para quantificar o efeito da termoterapia na população bacteriana. O título de Lxx foi determinado em folhas de plantas de duas variedades de cana-deaçúcar oriundas de toletes tratados termicamente ou não aos 90 dias após o plantio. O título inicial presente nos colmos utilizados como fonte de toletes interferiu na eficiência do tratamento, que reduziu a população bacteriana somente no ensaio onde os colmos apresentaram elevado título bacteriano inicial. Além disso, 70 e 55% das plantas oriundas de toletes tratados termicamente no primeiro e no segundo ensaio, respectivamente, apresentavam células de Lxx, porém em menores quantidades quando comparadas a plantas oriundas de toletes não tratados. Com isso, conclui-se que Lxx é transmitida em elevada frequência via sementes e a termoterapia, embora não tenha efeito erradicante pronunciado, é capaz de reduzir o título bacteriano no tecido vegetal.Due to its multiple uses, sugarcane (Saccharum spp.) is widely cultivated in more than 127 tropical and subtropical countries. In Brazil, sugarcane cropping has increased in recent years because of the growing demand for biofuels. However, the production of healthy seedlings tends to be a limiting factor to this increase, since contaminated setts transmit many diseases. Among these, the Ratoon Stunting Disease (RSD) is a major disease caused by the fastidious bacterium Leifsonia xyli subsp. xyli (Lxx) (KAO and DAMANN, 1980). According to the literature, the transmission of Lxx occurs through contaminated setts and/or cutting tools, but there are no reports of its transmission by seeds. Therefore, its control is based on the principle of exclusion using heat-treated setts as source of propagative material. However, the quantitative effect of the treatment on the bacterial titer is not known, since reports of thermotherapy efficiency are based on qualitative assessments. This study aimed to evaluate the transmission of Leifsonia xyli subsp. xyli by seeds and to quantify the effect of thermotherapy on Lxx in two sugarcane varieties using real-time quantitative PCR. In the test of disease transmission by seeds, the bacterium was detected in high incidences (>87%) in 90-day old plants generated from seeds collected from infected plants in quantities exceeding 51 cells per 100ng of total DNA of seeds. In addition, at 270 days after planting, the bacterium was isolated from six plants. The colony identity was confirmed by conventional PCR using Lxx-specific primers. Two trials were conducted to quantify thermotherapy effect on bacterial population. Lxx titers were determined 90 days after planting in plant leaves of two sugarcane varieties originated from setts subjected or not to heat-treatment. The initial bacterial titer in the stalks affected the treatment efficiency, which significantly reduced Lxx titers only in the trial where the stalks presented high initial bacterial levels. Moreover, 70 and 55% of the plants originated from heat-treated setts in the first and second tests, respectively, were infected with Lxx, cells; however, in smaller amounts compared to plants from untreated setts. Thus, it is concluded that Lxx is transmitted in high frequency by seeds and that the thermo treatment, although not efficient as an eradicating treatment, reduces the bacterial population in the plant tissue.
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Miranda, Cristiana Libardi. "Expressão diferencial de genes em células foliculares de suínos." Universidade Federal de Viçosa, 2007. http://locus.ufv.br/handle/123456789/4821.
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Among the productive traits, the number of the piglets is essential for success in swine production. The ovulation rate is one of the key factors that determine the litter size in pigs and the study of gene expression in the ovary follicles, more precisely in the follicular cells, might cause a big advance in the comprehension of this characteristic. Thus, the objective in this work was to identify the differential expression of the STAR (Steroidogenic Acute Regulator-STAR), GATA (GATA binding protein 4), PGF2α (Prostaglandin F2α), P4R (Progesterone Receptor), FSHR (Follicle Stimulating Hormone Receptor) and CYP19 (Cytochrome Aromatase P450) genes in follicular cells, colleted during the follicular phase in the estrous cycle of three sows with high and three sows with low litter size which comes from a tricross of the Landrace, Large White and Pietrain breeds, throughout the semi quantitative real time PCR (qPCR). In the hyperprolific sows, analyzed in this study, the average of the number of the piglets were 8,51 and for the hypoprolific sows the average was 12,03. For examination if any one related genes above were affecting the ovulation rate in the prolificity rate in the animals studied, the total RNA of the follicular liquid was extracted and than was made two pools with the total RNA of the hypo and hyperprolific sows groups. The total RNA of each pool was used for the first strand cDNA synthesis. The PCR reactions were accomplished using as an endogenous control the β-Actina gene. The primers were generated by sequences in the Pig ESTs data base. The major expression difference was observed for P4R gene, who express 7,73 turns mostly in the animals with low prolificity. The PGF2α gene displayed differential expression of 2,84 times as a large in the hypoprolific animals. The STAR gene expression, that codes for the protein regulating hormone steroids synthesis, was 2.32 turns mostly in the animals with low prolificity sows. The GATA gene expression was 2.31 times as a large in the hypoprolific sows. This gene codes for the GATA binding protein 4 belonging a transcription group factors that are responsible for too many gene expression and diverse cell differentiation types. The CYP19 gene expression taken the enzyme production that is the key factor in the estrogens biosynthesis, the Cytochrome Aromatase P450 (P450aro), for this gene was observed the relative expression of the 2.30 turns mostly in the sows with low prolificity. The FSHR gene expression hasn t achieved the threshold difference established in this study (1.5 times).The STAR, GATA, PGF2α, FSHR, P4R and CYP19 gene expression in the follicular cells has enabled identify difference of expression between sows with low and high prolificity, where the largest difference of expression in relation to the studied genes was verify in the hypoprolific sows, except for the FSHR gene that wasn t considerate differentially express in this work. With views the best agreement about the reproductive phenotypes in swine, it s necessary yet, studies to examine the expression in these and other genes related to female reproductive cycle, in as much as gene expression is dependent, in the midst of another factors, the stage of development of the tissue and the animal age.
Dentre as características produtivas, o número de leitões é fundamental para o sucesso na produção de suínos. A taxa de ovulação é um dos principais fatores que determinam o tamanho da leitegada. Deste modo, o estudo da expressão gênica nos folículos ovarianos, mais precisamente nas células foliculares, pode causar grande avanço no entendimento desta característica. Portanto o presente trabalho foi realizado com o objetivo de identificar a expressão diferencial dos genes STAR (Proteína esteroidogênica regulatória aguda), GATA (Proteína de ligação GATA-4), PGF2α (Prostaglandina F2α), P4R (Receptor de progesterona 4), FSHR (Receptor do hormônio folículo estimulante) e CYP19 (Citocromo aromatase P450) em células foliculares, coletadas durante a fase folicular do ciclo estral de três fêmeas suínas de alta e três de baixa prolificidade, provenientes de um tricross das raças Landrace, Large White e Pietrain, por meio da metodologia de PCR quantitativo em tempo real (qPCR). Nas fêmeas com alta prolificidade, utilizadas neste estudo, a média do número de leitões por leitegada foi 8,51 enquanto para as fêmeas com baixa prolificidade foi de 12,03. Para examinar se algum dos genes relacionados acima afetava as taxas de prolicifidade nos animais deste estudo, o RNA total do líquido folicular foi extraído e, em seguida, fez-se dois pools com o RNA total, sendo um do grupo de fêmeas com alta prolificidade e o outro do grupo com baixa prolificidade. O RNA total de cada um dos pools foi usado na confecção da primeira fita de cDNA. As reações de qPCR foram realizadas usando-se o gene β-Actina como controle endógeno. Os primers foram gerados a partir de seqüências obtidas nos bancos de ESTs de suínos. A maior diferença de expressão foi observada para o gene P4R, que foi expresso 7,73 vezes a mais nos animais com baixa prolificidade. O gene PGF2α apresentou expressão diferencial de 2,84 vezes a mais nas fêmeas hipoprolíficas. A expressão do gene STAR, que codifica para uma proteína reguladora da síntese de hormônios esteróides, foi 2,32 vezes maior nos animais com baixa prolificidade. A expressão do gene GATA foi 2,31 vezes maior nas fêmeas hipoprolíficas. Esse gene codifica para a proteína de ligação GATA-4, pertencente a um grupo de fatores de transcrição responsáveis pela expressão de vários genes e a diferenciação de diversos tipos celulares. A expressão do CYP19 leva à produção de uma enzima chave na biossíntese de estrogênio, a Citocromo aromatase P450 (P450aro), sendo que, para este gene, foi observada uma expressão relativa de 2,30 vezes a mais nas fêmeas de baixa prolificidade. O gene FSHR não atingiu o limiar de diferença de expressão, estabelecido neste estudo (1,5 vezes). A expressão dos genes STAR, GATA, PGF2α, FSHR, P4R e CYP19 nas células foliculares possibilitou a identificação de diferenças de expressão entre as fêmeas com alta e baixa prolificidade, sendo que a maior diferença de expressão para os genes estudados foi verificada nos animais de baixa prolificidade, exceto para o gene FSHR, que não foi considerado diferencialmente expresso nesse trabalho. Com vistas ao melhor entendimento dos fenótipos reprodutivos em suínos, é necessário que estudos sejam conduzifos no sentido de examinar a expressão desses e de outros genes relacionados, durante todo do ciclo reprodutivo das fêmeas, pois, a expressão de um gene depende, dentre outros fatores, do estádio do desenvolvimento do tecido e da idade do animal.
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Souza, William Marciel de. "Estudo evolutivo dos hantavírus e desenvolvimento de uma RT-PCR quantitativa em tempo real para detecção do vírus Araraquara." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/17/17147/tde-10042013-123852/.
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O gênero Hantavírus está incluído na família Bunyaviridae que são vírus emergentes associados a roedores que podem infectar o homem causando graves doenças. Nas Américas, os Hantavírus causam uma síndrome pulmonar e cardiovascular (SPCVH) com alta letalidade. Cerca de 1600 casos de SPCVH já foram notificados no Brasil causando mais de 600 óbitos. Sete espécies de Hantavírus são conhecidas no Brasil incluindo o vírus Araraquara que circula nas regiões de cerrado do país associado ao roedor Necromys lasiurus. Para o desenvolvimento de uma RT-PCR em tempo real para detecção e quantificação de Hantavírus, mostramos as etapas para o desenvolvimento de uma one-step RT-PCR em tempo real SYBR Green I para Hantavírus Araraquara que se mostrou específica para o gênero e capaz de detectar até 10 cópias por mL de RNA viral na amostra. Além disso, realizamos um estudo filogenético utilizando algoritmos bayesianos, com 190 sequências completas do gene da nucleoproteína, oriundas de 30 países durante um período de 25 anos (1985-2010) que encontravam-se disponíveis no GenBank (NCBI). Baseando-se em uma taxa média de 6.8 x 10-4 (2.5 x 10-4 - 1 x 10-3) substituições nucleotídicas por sítio/ano, foi possível inferir que os Hantavírus teriam aproximadamente 1917 anos. O processo de dispersão dos Hantavírus pelo mundo teria ocorrido há aproximadamente 500 anos, e a introdução destes vírus nas Américas teria ocorrido há 549 anos (95% HPD 1555-341 anos), via América Central ou México, originando os Hantavírus adaptados aos roedores da subfamília Neotominae, e pelo Brasil surgindo há 406 anos (95% HPD 1150-250 anos) os Hantavírus associados a roedores da subfamília Sigmodontinae, e posteriormente dispersaram para todo o continente sul-americano. O trabalho contribui de forma relevante para o diagnóstico das infecções por Hantavírus com a one-step RT-PCR em tempo real SYBR Green I e também, contribui para o entendimento da filogenia e história destes vírus, oferecendo subsídios ao entendimento sobre como teria ocorrido o espalhamento dos Hantavírus pelo mundo.The genus Hantavirus is included in the family Bunyaviridae are viruses emerging carried by rodents, which can infect humans causing serious illness. In the Americas, the Hantavirus causing a pulmonary syndrome (HPS) with high lethality. About 1,600 cases of HPS have been reported in Brazil, cause over 1600 deaths. Seven species of Hantavirus are known in Brazil, including Araraquara virus circulating in Cerrado regions (or Savannah regions) of the related in rodents Necromys lasiurus. The development of a real-time RT-PCR for detection and quantitation of Araraquara virus, here we show the steps for developing a one-step SYBR Green real-time RT-PCR for virus Araraquara which proved to be specific for the genus and capable of detecting up to 10 copies of viral RNA per ml in the sample. Furthemore, we performed a phylogenetic analysis using Bayesian algorithms, with 190 complete sequences of the nucleoprotein gene, originating from 30 countries over a 25 year period (1985-2010) that were available in GenBank (NCBI). Based on an average rate of 6.8 x 10-4 (2.5 x 10-4 - 1 x 10-3) nucleotide substitutions per site/year, it was possible to infer that the Hantavirus would be about 1917 years old. The Hantavirus spreading in the world have occurred for nearly 500 years, and the introduction of these viruses have occurred in the Americas 549 years ago (95 years% HPD 1555-341) bye Central America or Mexico, causing the Hantavirus adapted to rodents subfamily Neotominae, and Brazil emerged 406 years ago (95% HPD 1150-250 years) the Hantavirus associated with rodents subfamily Sigmodontinae, and subsequently disseminated to South America. The work contributes significantly to the diagnosis of Hantavirus infections with one-step SYBR Green real-time RT-PCR and also contributes to an understanding of the phylogeny and evolutionary history of these viruses, offering subsidies have occurred understanding of how the Hantavirus spread of the worldwide.
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ALBUQUERQUE, Yvana Maria Maia de. "Reação em cadeia da polimerase em tempo real quantitativa (QPCR) para diagnóstico da tuberculos pulmonar em escarro de pacientes com HIV/aids." Universidade Federal de Pernambuco, 2012. https://repositorio.ufpe.br/handle/123456789/18503.
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O diagnóstico da tuberculose apresenta dificuldades em pacientes soropositivos para o vírus da imunodeficiência humana (HIV). Os doentes coinfectados HIV/M.tuberculosis(MTB)nos estágios mais avançados de imunocomprometimento apresentam manifestações clínicas atípicas e o exame direto, rotineiramente utilizado, tem baixa sensibilidade. A cultura apesar de ter maior sensibilidade fornece resultados tardios. Evidencia-se nesta população a necessidade de testes diagnósticos mais eficientes. A tese será apresentada no formato de dois artigos científicos. No primeiro, estudou-se a utilidade da Reação em Cadeia da Polimerase em tempo real quantitativa (qPCR) para diagnóstico da tuberculose pulmonar em escarro de pacientes com HIV/aids. No segundo, descreveu-se as alterações radiográficas do tórax de pacientes com tuberculose pulmonar confirmado por cultura de escarro. Foram incluídos no estudo 140 pacientes com HIV/aids e suspeita clínica de tuberculose pulmonar, atendidos no período de agostode 2009 a janeiro de 2012, em dois hospitais de referência para atendimento de pacientes infectados pelo HIV em Recife-PE. Coletou-se uma amostra de escarro de cada paciente, e caso não houvesse escarro suficiente, realizou-se nebulização com solução salina para indução do escarro. O padrão ouro do estudo foi à cultura realizada em meios Löwenstein-Jensen e 7H9. A cultura e a qPCR para tuberculose foram realizadas em laboratório privado situado no Recife. Dos 140 pacientes em 47 (33,6%), diagnosticou-se tuberculose pulmonar pelo padrão ouro. A sensibilidade, especificidade e acurácia da qPCR foram respectivamente 87,2%, 98,9% e 95%. Foram realizados exames radiográficos do tórax em 42 pacientes coinfectados com cultura de escarro positiva, que foram avaliados por dois radiologistas experientes. A alteração radiológica isolada mais frequente observada foi a consolidação parenquimatosa, que acometeu seis (14,3%) dos pacientes, seguida pelo infiltrado intersticial e micronodular difuso, além da associação infiltrado e consolidação. Concluiu-se que a qPCR realizada no escarro de pacientes coinfectados HIV/MTB apresentou boa sensibilidade, especificidade e acurácia, sendo útil no diagnóstico de tuberculose pulmonar nesses pacientes. Com relação aos achados radiográficos de tórax, estes demonstraram ser de pouco auxílio no diagnóstico da tuberculose pulmonar nos coinfectados, exceto quando o padrão cavidade e infiltrado micronodular difuso estão presentes.
Tuberculosis’ diagnosis is difficult in HIV soropositivepatients. The patients co-infected HIV/M.tuberculosis(MTB) in severe immune deficiency stage present atypical clinical manifestation and direct sputum smear, usually used, shows low sensitivity.The culture, despite better sensitivity, obtains later results. The thesis will be presented in form of two articles. In the first, it has studied the utility of quantitative real time PCR for tuberculosis’ diagnosis among AIDS patients’ sputum smear. In the second, it has been described the major thoracic radiographic alterations among AIDS patients’ and pulmonary tuberculosis confirmed by sputum culture. A total of 140 HIV/AIDS patients were included in the study, with clinical suspicion of pulmonary tuberculosis, from August 2009 to January 2012, were attended at two referral hospitals for HIV/AIDS in Recife-PE. A sputum sample was collected from each patient, and if they were unable to produce spontaneous sputum, they were saline nebulized, for sputum induction. The culture, in Löwenstein-Jensen solid medium and 7H9, was the gold standard. The culture and qPCR for tuberculosis have been done in a private laboratory in Recife-PE. From all the 140 studied patients, 47 (33,6%) pulmonary tuberculosis was diagnosis by gold standard. The sensitivity, specificity and accuracy were 87,7%, 98,9% and 95% respectively. There were evaluated chest X-rays from 42 co-infected HIV/MTB patients with positive culture by two experienced radiologists, the most common radiological alteration was parenchymal consolidation, encountered in six (14,3%) patients, followed by interstitial infiltrate, difuse micronodular (military) pattern an association between interstitial infiltrate and parenchymal consolidation. It was concluded that qPCR, has given a good sensitivity, specificity and accuracy and it can be recommender for use in the management of HIV/AIDS patients. However, thoracic radiographic findings were not specific and thorax RX is not sufficient initself to establish a pulmonary tuberculosis diagnosis in co-infected HIV/MTB patients, except when cavity and micronodular pattern are presented.
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Swatoch, Phillipp Christopher. "Etablierung und Evaluierung einer real-time RT-PCR zur quantitativen Analyse der Genexpression von 7 humanen Zytokinen." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975528513.
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Julião, Fred da Silva. "Uso de método de biologia molecular quantitativo (PCR real-time) na avaliação de reservatórios para leishmaniose visceral." reponame:Repositório Institucional da FIOCRUZ, 2011. https://www.arca.fiocruz.br/handle/icict/4206.
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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
A leishmaniose visceral (LV) é uma zooantroponose sistêmica de importância reconhecida em saúde pública, 90% dos casos no novo mundo são oriundos do Brasil. Os cães domésticos e as raposas são considerados como os principais reservatórios. A persistência da Leishmania em áreas endêmicas e o insucesso das medidas de prevenção, dirigidas exclusivamente ao reservatório canino, sugerem que outros animais podem ter importância na manutenção do ciclo de transmissão da LV. OBJETIVO: Avaliar potenciais reservatórios para LV numa área endêmica, utilizando método de biologia molecular quantitativo (PCR real-time). MÉTODOS: Foram estudados animais domésticos (bovinos, equídeos, caprinos e ovinos) e animais silvestres (marsupial), no município de Salinas da Margarida, Bahia, de 2007 a 2009. Todos os animais domésticos de produção mantidos e/ou pernoitando nas áreas urbanas do município foram incluídos. Os marsupiais foram capturados com armadilha animal modelo Tomahawk colocadas no peridomicílio das residências onde ocorreram casos de LV humana e/ou canina na localidade de Encarnação. Nos animais domésticos de produção foi coletado apenas amostra de sangue periférico e nos marsupiais, além de sangue, foi obtida uma amostra de pele através de biópsia da orelha. Em todas as amostras foi realizado PCR real-time para investigar a presença de DNA do parasito e estimar a carga parasitária. Os primers e sondas utilizados foram selecionados em gene SSU rRNA, que aparece 160 vezes no genoma de Leishmania spp. e é altamente conservado entre as espécies de Leishmania. RESULTADOS: No total, foram avaliados 80 animais domésticos (20 bovinos, 33 equídeos, 20 caprinos e 7 ovinos) e 103 marsupiais, todos da espécie Didelphis albiventris. Cinco bovinos foram positivos no teste de PCR real-time com carga parasitária variando de 12,7 a 183,5 parasitos/mL. Apenas um marsupial apresentou amostra de sangue positiva (6,0 parasitos/mL). Todos os demais animais testaram negativo. CONCLUSÃO: A técnica de PCR real-time pode ser uma ferramenta útil para avaliar o papel de potenciais reservatórios domésticos e silvestres para LV. A execução do PCR real-time é menos trabalhosa e mais prática do que a realização do teste de xenodiagnóstico, além disso, ela poder ser automatizada, permitindo a análise de grande número de amostras em estudos epidemiológicos. A detecção de carga parasitária de Leishmania em sangue de bovinos, em quantidade comparável à encontrada em cães, sugere que eles podem ser reservatórios para LV. A relativa abundância de bovinos nas áreas endêmicas para LV, assim como as evidências da preferência alimentar do vetor por estes animais, ressaltam a importância do papel que os bovinos podem ter na transmissão da LV. Mais trabalhos são necessários para elucidar estas questões.
Visceral leishmaniasis (VL) is a systemic zooantroponose of public health relevance, 90% of cases in the new world are from Brazil. Domestic dogs and foxes are considered the main reservoirs. The persistence of Leishmania in endemic areas and the failure of preventive measures, directed exclusively to the canine reservoir, suggest that other animals may be important in maintaining the transmission cycle of Leishmania. OBJECTIVE: To investigate potential reservoirs for VL in an endemic area, using molecular biology quantitative method (real-time PCR). METHODS: We studied domestic animals (cattle, horses, goats and sheeps) and wildlife (marsupial), in the city of Salinas da Margarida, Bahia, from 2007 to 2009. All livestock animals maintained and/or staying overnight in the urban areas of the municipality were included. The marsupials were captured with Tomahawk model animal traps placed outside the home of homes where there were human and/or dog VL cases in the locality of the Encarnação. In livestock animals, we collected only a sample of peripheral blood and in marsupials, in addition to blood we also collected a sample of ear skin biopsy. In all samples we carried out real-time PCR to detect the presence of parasite DNA and to estimate the parasite load. The primers and probes used were selected on SSU rRNA gene, which appears 160 times in the genome of Leishmania spp. and is highly conserved among species of Leishmania. RESULTS: In total, 80 livestock animals were evaluated (20 cattle, 33 horses, 20 goats and 7 sheep), and 103 marsupials, all Didelphis albiventris. Five cattle were positive by real-time PCR with parasite load ranging from 12.7 to 183.5 parasites/mL. Only one marsupial had a positive blood sample (6.0 parasites/mL). All other animals tested negative. CONCLUSION: The real-time PCR technique can be a useful tool for assessing the potential role of domestic and wild reservoir for LV. The implementation of real-time PCR is less laborious and more practical than the test of xenodiagnosis, in addition, it can be automated, allowing for the analysis of large number of samples in epidemiological studies. Detection of Leishmania parasite load in the blood of cattle, in an amount comparable to that found in dogs, suggests that they may be reservoirs for VL. The relative abundance of cattle in endemic areas for VL, as well as evidence of vector feeding preference for these animals, highlight the important role that cattle may exert in the transmission of VL. More research is needed to clarify these issues.
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Chao, Ying Sheng. "Development of quantitative real time PCR to assess human brain microvascular endothelial cell susceptibility to HIV-1 infection." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1450192.
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Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed April 7, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 64-70).
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Wildemann, Paula. "Pesquisa de Yersinia Enterocolitica patogênica em tonsilas de suínos ao abate em Santa Catarina." Universidade do Estado de Santa Catarina, 2016. http://tede.udesc.br/handle/handle/2545.
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Capes
Yersinina enterocolitica is a Gram-negative bacteria with zoonotic potential. It is associated with the occurrence of enteric diseases in humans. Pigs are considered the main source of Y. enterocolitica and the bacteria is mainly found in the pig’s palatine tonsils. The objective of this study was to evaluate the occurrence of pathogenic Y. enterocolitica in palatine tonsils of healthy pigs from Santa Catarina, during the slaughter process. In order to achieve this goal, a multiplex PCR technique was performed so as to detect the presence of virulence genes (ail, yadA and virF). This technique was compared to quantitative real time PCR (qPCR), only for the ail gene. Palatine tonsils were randomly collected from 400 pigs from four federally inspected slaughterhouses of the state of Santa Catarina. One positive sample was found for the three studied virulence genes, which were confirmed by DNA sequencing. The analysis of partial sequences of the three virulence genes identified three unique amino acid changes, one in the virF gene and two in YadA gene. This sample had 11.058.398 molecules/μL detected by qPCR. By comparing the two techniques, qPCR was 100 times more sensitive than standard PCR. This result shows low occurrence of pathogenic Y. enterocolitica in healthy pigs from federally inspected slaughterhouses in Santa Catarina
Yersinia enterocolitica é uma bactéria Gram-negativa emergente que possui potencial zoonótico e está associada a quadros de infecção alimentar em humanos. Os suínos são considerados o principal reservatório de Y. enterocolitica, abrigando-a principalmente nas tonsilas. Tendo em vista a carne suína como uma das mais consumidas no mundo e a importância deste agente zoonótico, o objetivo deste trabalho foi avaliar a ocorrência de Y. enterocolitica patogênica em tonsila de suínos no momento do abate no estado de Santa Catarina. Para isto, foi utilizada uma PCR convencional multiplex que detecta a presença de genes de virulência (ail, yadA e virF) e comparou-se esta técnica com a PCR quantitativa em tempo real (qPCR), somente para o gene ail. Foram coletadas aleatoriamente tonsilas de 400 suínos provenientes de quatro frigoríficos com inspeção federal em diferentes regiões do estado. Foi realizado o sequenciamento do DNA dos genes amplificados das amostras positivas na cPCR e posteriormente foi feita a análise filogenética. Apenas uma amostra foi positiva para os três genes pesquisados na PCR convencional, os quais foram confirmados por sequenciamento. A análise das sequências parciais dos três genes de virulência identificou três mudanças de aminoácidos exclusivas, sendo uma no gene virF e duas no gene yadA. Na qPCR esta amostra apresentou 11.058.398 moléculas/μL. Ao comparar as duas técnicas, a qPCR foi 100 vezes mais sensível que a PCR convencional. Isso demonstra uma baixa ocorrência de Y. enterocolitica patogênica em suínos sadios ao abate em frigoríficos com inspeção federal em Santa Catarina
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Walter, Christina. "Quantifizierung des postmortalen RNA-Status im Gehirn mittels Real-time-PCR : Ein Beitrag zur Bestimmung der Leichenliegezeit." Doctoral thesis, kostenfrei, 2008. http://nbn-resolving.de/urn/resolver.pl?urn=nbn:de:bvb:20-opus-28570.
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Eiderbrant, Kristina. "Development of quantitative PCR methods for diagnosis of bacterial vaginosis and vaginal yeast infection." Thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-68269.
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Vaginitis is a vaginal infection which affects many women all over the world. The disorder is characterized by an infection of the vaginal area which can cause problems like abnormal vaginal discharge, itching and redness. The two most common causes of vaginitis are bacterial vaginosis and Candida vaginitis. The prevalence of bacterial vaginosis in Sweden is around 10-20 % and approximately 75 % of all women will once in their lifetime suffer from vaginal yeast infection. The clinical symptoms of vaginal infections are not specific and the diagnosis methods of bacterial vaginosis and Candida vaginitis are subjective and depended on the acuity of the clinician. Due to the lack of standardized and objective diagnostic tools, misdiagnosis and consequently incorrect treatment may occur. As vaginal infections and symptoms impact greatly of women´s quality of life and vaginitis have been associated with serious public health consequences, it is essential to diagnose and treat the conditions correctly. Hence, there is a great need of better methods of diagnosing these conditions. The aim of this master thesis was to develop quantitative species-specific real-time PCR assays to use in diagnosing the two most common causes of vaginitis i.e. bacterial vaginosis and Candida vaginitis. Potential markers for bacterial vaginosis (Atopobium vaginae, BVAB2, Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Megasphaera type 1, Megasphaera type 2, Mobiluncus curtisii, Mobiluncus mulieris and Leptotrichia/Sneathia species) and Candida vaginitis (Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis) were chosen. Primers and probes were designed and tested on reference strains and vaginal samples. Single- and multiplex PCR reactions were successfully optimized with the designed oligonucleotides. Furthermore, standard curves with excellent linearity were created and covered more than five orders of magnitude. These developed quantitative species-specific real-time PCR assays will, in a prospective medical validation, quantify 300 vaginal samples from women visiting the RFSU Clinic in Stockholm.
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Dölken, Marc Thorsten. "Etablierung einer quantitativen, allel-spezifischen "real-time" PCR für rearrangierte Immunglobulingene des IgH-Locus maligner B-Zellen-Lymphome." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=972354247.
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Tschürtz, Florian [Verfasser]. "Monitoring der Minimalen Resterkrankung in der NPM1 mutierten Akuten Myeloischen Leukämie mittels quantitativer Real-Time PCR / Florian Tschürtz." Ulm : Universität Ulm. Medizinische Fakultät, 2014. http://d-nb.info/1052269842/34.
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Diaz, Cruz Maria Araceli. "Quantitative detection of Sclerotinia sclerotiorum and prediction of stem rot rape seed plants disease by using real time PCR." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-11550.
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Schulze, Felix, Deeksha Malhan, Khassawna Thaqif El, Christian Heiss, Anja Seckinger, Dirk Hose, and Angela Rösen-Wolff. "A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-232280.
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BACKGROUND:
In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability.
METHODS:
Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes.
RESULTS:
We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples.
CONCLUSION:
This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.
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Schulze, Felix, Deeksha Malhan, Khassawna Thaqif El, Christian Heiss, Anja Seckinger, Dirk Hose, and Angela Rösen-Wolff. "A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model." BioMed Central, 2017. https://tud.qucosa.de/id/qucosa%3A30735.
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BACKGROUND:
In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability.
METHODS:
Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes.
RESULTS:
We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples.
CONCLUSION:
This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.
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Tsuchido, Yasuhiro. "Real-time quantitative PCR analysis of endoscopic biopsies for diagnosing CMV gastrointestinal disease in non-HIV immunocompromised patients: a diagnostic accuracy study." Kyoto University, 2019. http://hdl.handle.net/2433/242399.
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Unglaube, Sandra. "In-vitro-Untersuchungen zur quantitativen Vitalitätsbeurteilung von C. parvum-Oozysten." Doctoral thesis, Universitätsbibliothek Leipzig, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:15-20091027-131947-1.
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Die Arbeit hatte zum Ziel, ein In-vitro-Infektionsmodell für den protozoären Durchfallerreger Cryptosporidium parvum zu optimieren und dahingehend zu testen, ob es für eine quantitative Beurteilung der Infektiosität von Kryptosporidienoozysten eingesetzt werden kann. Die verwendeten Oozysten wurden zuvor im Zuge einer Passagierung im Kalb vermehrt, aus dem Kot isoliert, aufgereinigt und zur Infektion einer humanen ileocaecalen Adenokarzinomzelllinie (HCT-8) verwendet Die Kultivierung erfolgte über 48 Stunden in Mikrotiterplatten mit jeweils 24 Kavitäten. Die DNA infizierter Zellen und nichtinfizierter Kontrollen wurde anschließend isoliert und die parasitenspezifische DNA in der real-time PCR quantifiziert. Die gewählten Primer-Sonden-Kombinationen erlaubten eine spezifische Amplifikation der Erreger-DNA. In der Optimierung wurden das Brilliant®QPCR Core Reagent Kit, der ABsoluteTMQPCR sowie zwei verschiedene Oligonukleotidkombinationen untersucht. Durch die Klonierung einer Sequenz im Target-Gen und die Herstellung einer Titrationsreihe aus dieser klonierten DNA gelang es, den für die Vergleichbarkeit unerlässlichen homogenen Standard zu gewinnen. Der In-vitro-Vitalitätsassay wurde außerdem auf seine praktische Anwendbarkeit hin geprüft. Es wurde einerseits eine Desinfektionsmittelprüfung mit Chlorokresol (Neopredisan®135-E), andererseits ein Versuch zur thermischen Inaktivierung, beide unter Nutzung dreier verschiedener C. parvum-Chargen (LE-06-Cp-05/0, LE-07-Cp-05/2 vom Isolat A, LE-06-Cp-05/2 vom Isolat B), vollzogen. Die Überbewertung der Infektiosität der Oozysten durch die Betrachtung der Exzystierung konnte anhand der parallel zur DNA-Quantifizierung ermittelten Exzystierungsraten gezeigt werden. Die Exzystierungshemmung lag in jedem Versuch deutlich unter den in der real-time PCR berechneten Inaktivierungsraten. Je nach verwendeter Oozystencharge lieferte die Desinfektion mit 4 % Neopredisan®135-E Inaktivierungsraten, die zwischen 90 und 100 % bei einstündiger Einwirkzeit lagen. Mit steigender Dauer der Inkubation stieg erwartungsgemäß auch der Grad der Inaktivierung. Die Anwendung der 1 %igen Verdünnung resultierte in einer deutlich gesteigerten Exzystierungsrate gegenüber der unbehandelten Kontrolle sowie in stark variierenden Inaktivierungsraten (24 - 91,5 %). Es konnte gezeigt werden, dass mit Neopredisan®135-E unter den gewählten Inkubationsbedingungen zwar eine gute, aber keine vollständige Inaktivierung der C. parvum-Oozysten erfolgt. Eine suboptimale Wirkung zeigte sich in einer hohen Varianz der Einzelmesswerte. Die Vitalitätsraten betrugen nach einstündiger Inkubation der Oozysten bei 38°C noch 100 %, nach 24 Stunden waren diese bereits auf 5 - 23 % abgesunken. Es scheint, als würden mesophile Verhältnisse die Exzystierung der Sporozoiten anregen und bei längerer Konditionierung eine Erschöpfung des Stoffwechsels der Entwicklungsstadien herbeiführen. Die Inaktivierungsrate bei 55°C lag zwischen 96 und 100 %. Bei thermophiler Konditionierung wurde in drei von sieben Fällen, nach der Inkubation in Neopredisan®135-E nur in einer der sieben Untersuchungen ein vollständiger Vitalitätsverlust beobachtet. Die vorgestellte Methode erwies sich als gut reproduzierbar, sensitiv und schnell. Die In-vitro-Kultivierung des Erregers C. parvum ließ sich mit der real-time PCR, welche eine absolute Quantifizierung erlaubte, gut in Einklang bringen. Die Verwendung der In-vitro-Kultur als lebendes System ließ eine gewisse Variabilität der Ergebnisse zwischen einzelnen Untersuchungen erwarten, die sich aber in einem akzeptablen Bereich bewegten. Eine weitere Optimierung im Sinne einer Sensitivitätssteigerung bei akzeptabler Störanfälligkeit und Variabilität ist anzustreben.
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Mackay, Ian M. "Investigations into the utility of real-time PCR for the detection, quantitation and characterisation of clinically relevant viruses /." St. Lucia, Qld, 2003. http://adt.library.uq.edu.au/public/adt-QU20031120.155312/index.html.
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