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Littérature scientifique sur le sujet « Real-Time quantitative PCRs »

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Publié le 11 mai 2023

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Articles de revues sur le sujet "Real-Time quantitative PCRs"

Blanda, Valeria, Rosalia D’Agostino, Elisabetta Giudice, Kety Randazzo, Francesco La Russa, Sara Villari, Stefano Vullo et Alessandra Torina. « New Real-Time PCRs to Differentiate Rickettsia spp. and Rickettsia conorii ». Molecules 25, n^o 19 (27 septembre 2020) : 4431. http://dx.doi.org/10.3390/molecules25194431.

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Rickettsia species are an important cause of emerging infectious diseases in people and animals, and rickettsiosis is one of the oldest known vector-borne diseases. Laboratory diagnosis of Rickettsia is complex and time-consuming. This study was aimed at developing two quantitative real-time PCRs targeting ompB and ompA genes for the detection, respectively, of Rickettsia spp. and R. conorii DNA. Primers were designed following an analysis of Rickettsia gene sequences. The assays were optimized using SYBR Green and TaqMan methods and tested for sensitivity and specificity. This study allowed the development of powerful diagnostic methods, able to detect and quantify Rickettsia spp. DNA and differentiate R. conorii species.

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Joly, Philippe, Pierre-Alain Falconnet, Janine André, Nicole Weill, Monique Reyrolle, François Vandenesch, Max Maurin, Jerome Etienne et Sophie Jarraud. « Quantitative Real-Time Legionella PCR for Environmental Water Samples : Data Interpretation ». Applied and Environmental Microbiology 72, n^o 4 (avril 2006) : 2801–8. http://dx.doi.org/10.1128/aem.72.4.2801-2808.2006.

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ABSTRACT Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >103 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.

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Ruszova, E., M. Chmelarova, M. Senkerikova et S. Stefackova. « Duplex Methylation-Specific Semi-Quantitative Real-Time PCR for Cost-Effective & ; Time-Efficient Diagnostic Screening of Chromosome 15 and 14 Imprinted Regions ». Acta Medica Martiniana 15, n^o 3 (1 décembre 2015) : 5–12. http://dx.doi.org/10.1515/acm-2015-0012.

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AbstractPurpose: Our goal was to develop two-tier strategy based onin house-designed methylation specific-duplex polymerase chain reactions (MS-PCRs) that could serve as a relatively simple, cost effective, time efficient approach for molecular screening of imprinted regions on chromosomes 15 and 14.Patients and methods: Patients were referred to examination during infancy due to hypotonia and motor development delay. Duplex MS-PCRs were performed that enabled detection of methylated/unmethylated DNA inNDNandMEG3CpG islands via plurality of detection channels on PCR instrument Rotor Gene 6000.Results and discussion: Both, copy number variations as well as methylation changes, were revealed by ourin house-designed methodology by focusingNDNgene. No imprinting aberrations were yet discovered inMEG3gene. Clinical features of the patients were compared. In agree with literature no typical facial features were observed in PWS patient with imprinting defect and AS UPD patient seems to have a relatively better development and language ability in comparison to deletional form of the disease.Conclusion: In conclusion we were able to establish new, throughput and robust diagnostic approach to PWS/AS.

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Papp, Stefanie, Jessica Rauch, Svenja Kuehl, Ulricke Richardt, Christian Keller et Anke Osterloh. « Comparative evaluation of two Rickettsia typhi-specific quantitative real-time PCRs for research and diagnostic purposes ». Medical Microbiology and Immunology 206, n^o 1 (1 octobre 2016) : 41–51. http://dx.doi.org/10.1007/s00430-016-0480-z.

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Lopotova, Tereza, Jana Moravcova, Vaclava Polivkova, Jaroslav Polak, Jiri Schwarz, Hana Klamova et Katerina Machova Polakova. « Expression of Four Major WT1 Splicing Variants in AML and CML : Development of Quantitative Real-Time PCRs and Preliminary Results in Patient Samples. » Blood 114, n^o 22 (20 novembre 2009) : 1631. http://dx.doi.org/10.1182/blood.v114.22.1631.1631.

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Abstract Abstract 1631 Poster Board I-657 WT1 expression has proved to be of prognostic significance in AML, MDS and according to our unpublished data also in CML patients. There are four major WT1 isoforms with distinct functions in cells. Splicing variants encoding the WT1 isoforms differ from each other by the presence or absence of both the exon 5 and KTS region in the exon 9 of WT1 gene. It was shown that exon 5 containing isoforms appear to cause cell cycle arrest. Therefore, differences in WT1 splicing variant ratios might be associated with prognosis and response to therapy. All currently used primer/probe systems quantify all major splicing variants as expression of total WT1. The aim of our study was to develop specific quantitative real-time PCRs (qRT-PCRs) for quantification of the four major WT1 splicing variants (+5+KTS; +5-KTS; -5+KTS; -5-KTS) at mRNA level and to apply these techniques for initial screening of WT1 splicing variants expression in AML and CML patients. We designed several sequence variants of two forward (specific for +/-5) and two reverse primers (specific for +/-KTS) and one common TaqMan probe. After in silico analyses, selected primers were subjected for testing with plasmids each carrying target sequence of the splicing variant. The qRT-PCRs were performed on RotorGene (Corbett Research). GUS was used as housekeeping gene; primers, probes and protocols were adopted from Gabert et al. (Leukemia 2003, 17:2318). Absolute quantification was applied for data evaluation (reported in percentage). Most of these tests focused on qRT-PCR specificity. The main test of quantification utility and specificity was performed by using 25 plasmid mixtures that simulate different ratios of particular variants (copies of each variant were in five different ratios to the remaining three variants; from 10/100000 to 100000/10 copies). Finally, total leukocytes from 45 AML and 13 CML patients at diagnosis were analysed to test system utility on real samples. The splicing ratio stability during the time was checked, because there were some specimens of peripheral blood not processed immediately but preserved at 4°C overnight. Our results showed high specificity and utility of all four qRT-PCRs. We were able to quantify up to 100 copies of each variant. Even in case that one of the variants was in minority compared to others in the plasmid mixture, expected number of plasmid copies was assessed by the qRT-PCR. Splicing variant ratio was found to be conserved, when the blood was preserved overnight in refrigerator. Observed differences fall within the ranges of the technical variability of the four qRT-PCRs (12-50%). In general, our preliminary data in patient samples showed the +5/-KTS variant as the most abundant in patients with CML and AML. Expression of the +5/-KTS and -5/+KTS highly varied among AML patients within the same FAB subtype. Low level of +5/+KTS variant (median 18% from total WT1) distinguished the high risk group patients (cytogenetic-based risk stratification) from the remaining groups (median 30% from total WT1). Our data also indicate that +KTS/-KTS variant expression levels independent of the presence of exon 5 may be of importance. While in low and intermediate risk groups +KTS and –KTS variants were expressed without significant differences, in the high risk group of patients the expression of –KTS variants was significantly higher. At the same time, the high risk group showed significantly decreased +5 variants levels (independent of the presence of KTS sequence). In conclusion, we have developed the specific qRT-PCRs detecting the four WT1 splicing variants on mRNA level. Further research is ongoing to confirm the aforementioned significance of different expression of the particular WT1 isoforms. Supported by MZOUHKT2005. Disclosures No relevant conflicts of interest to declare.

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Maksimov, Pavlo, Hannes Bergmann, Marion Wassermann, Thomas Romig, Bruno Gottstein, Adriano Casulli et Franz J. Conraths. « Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs ». Pathogens 9, n^o 10 (26 septembre 2020) : 791. http://dx.doi.org/10.3390/pathogens9100791.

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Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.

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Wall, Steven J., et Dylan R. Edwards. « Quantitative Reverse Transcription–Polymerase Chain Reaction (RT-PCR) : A Comparison of Primer-Dropping, Competitive, and Real-Time RT-PCRs ». Analytical Biochemistry 300, n^o 2 (janvier 2002) : 269–73. http://dx.doi.org/10.1006/abio.2001.5458.

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Gautam, Rashi, Slavica Mijatovic-Rustempasic, Mathew D. Esona, Ka Ian Tam, Osbourne Quaye et Michael D. Bowen. « One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples ». PeerJ 4 (11 janvier 2016) : e1560. http://dx.doi.org/10.7717/peerj.1560.

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Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

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Wen, Jing, Hong Yang, Kongjia Luo, Yi Hu, Xu Zhang, Geng Wang, Yuping Chen et al. « A three-gene expression signature model to predict neo-chemoradiotherapy response of esophageal squamous cell carcinomas. » Journal of Clinical Oncology 31, n^o 15_suppl (20 mai 2013) : e15135-e15135. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e15135.

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e15135 Background: Preoperative chemoradiotherapy (CRT) followed by surgery has been proved to improve survival in comparison with surgery alone. However, the outcomes of CRT are heterogeneous, and no clinical or pathological method could prediction CRT response. In this study, we aim to identify mRNA markers for ESCC CRT-response prediction. Methods: Gene expression analyses were performed on pretreatment cancer biopsies from 28 ESCCs who received neoadjuvant CRT and surgery. Surgical specimens were assessed for the pathological response to CRT. The identified differentially expressed genes were validated by real-time quantitative polymerase chain reaction (qPCR), based on which a classifying model was built up by Fisher’s linear discriminant analysis. The predictive power of this model was further assessed in another set of 32 ESCCs. Results: The profiling of the 28 ESCCs identified 10 differentially expressed genes with more than 2-fold changes between pathological complete responsers (pCRs) and less than pCRs (<pCRs), among which 6 genes (LIMCH1, SDPR, Clorf226, SLC9A9, GSTM3, and IGSF10) were down-regulated and 4 genes (MMP9, MMP1, MMP12 and OASL) up-regulated in pCRs versus <pCRs. A prediction model based on qPCR values of 3 genes was built up, Y=-10.388 - 0.343 × MMP1 + 0.642 × LIMCH1 + 0.921 × Clorf226 with a cut-off value of 0.607. It provided a predictive accuracy of 85.7% with leave-one-out cross-validation. Further, the predictive power of this model was validated in another set of 32 ESCCs, among which a predictive accuracy of 81.3% was achieved. Conclusions: The combination of three genes by qPCR identified by microarrays in our study provides possibility for ESCC CRT prediction, which will facilitate individualization of ESCC treatment. Further perspective validation in larger independent cohorts is warranted to fully assess the predictive power of this prediction model.

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Skubic, Lucijan, Lea Hošnjak, Vesna Breznik, Kristina Fujs Komloš, Boštjan Luzar et Mario Poljak. « An Improved Protocol for Comprehensive Etiological Characterization of Skin Warts and Determining Causative Human Papillomavirus Types in 128 Histologically Confirmed Common Warts ». Viruses 14, n^o 10 (15 octobre 2022) : 2266. http://dx.doi.org/10.3390/v14102266.

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Human papillomaviruses (HPVs) are etiologically associated with various benign and malignant neoplasms of cutaneous and mucosal epithelia. We describe an improved diagnostic protocol for comprehensive characterization of causative HPV types in common warts, in which broad-spectrum PCRs followed by Sanger sequencing, two previously described and seven newly developed type-specific quantitative real-time PCRs (qPCRs) coupled with the human beta-globin qPCR were used for: (i) diagnosis of HPV infection in warts; (ii) estimation of cellular viral loads of all HPV types detected; and (iii) determination of their etiological role in 128 histologically confirmed fresh-frozen common wart tissue samples. A total of 12 different causative HPV types were determined in 122/126 (96.8%) HPV-positive warts, with HPV27 being most prevalent (27.0%), followed by HPV57 (26.2%), HPV4 (15.1%), HPV2 (13.5%), and HPV65 (7.9%). The cellular viral loads of HPV4 and HPV65 were estimated for the first time in common warts and were significantly higher than the viral loads of HPV2, HPV27, and HPV57. In addition, we showed for the first time that HPV65 is etiologically associated with the development of common warts in significantly older patients than HPV27 and HPV57, whereas HPV4-induced warts were significantly smaller than warts caused by HPV2, HPV27, HPV57, and HPV65.

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Thèses sur le sujet "Real-Time quantitative PCRs"

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 globalité
Leptospirosis 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 et 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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Livres sur le sujet "Real-Time quantitative PCRs"

Biassoni, Roberto, et Alessandro Raso, dir. Quantitative Real-Time PCR. New York, NY : Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0733-5.

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Biassoni, Roberto, et Alessandro Raso, dir. Quantitative Real-Time PCR. New York, NY : Springer New York, 2020. http://dx.doi.org/10.1007/978-1-4939-9833-3.

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Quantitative real-time PCR in applied microbiology. Norfolk, UK : Caister Academic Press, 2012.

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Quantitative real-time PCR : Methods and protocols. New York : Humana Press, 2014.

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Biassoni, Roberto, et Alessandro Raso. Quantitative Real-Time PCR : Methods and Protocols. Springer New York, 2016.

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Biassoni, Roberto, et Alessandro Raso. Quantitative Real-Time PCR : Methods and Protocols. Springer New York, 2019.

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Biassoni, Roberto, et Alessandro Raso. Quantitative Real-Time PCR : Methods and Protocols. Springer New York, 2020.

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Taberlet, Pierre, Aurélie Bonin, Lucie Zinger et Eric Coissac. Single-species detection. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198767220.003.0009.

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Chapter 9 “Single-species detection” deals with the practical aspects of detecting a single and predefined taxon with eDNA, with a particular focus on the use of quantitative PCR (qPCR) for this purpose. After presenting how single-species detection has been implemented in a few seminal studies, it details the principles underlying qPCR. More specifically, it describes the typical qPCR amplification curve and the different systems (SYBR green and TaqMan probe assays) available to record amplicon accumulation in real time via fluorescence measurements. Chapter 9 also explains how the initial number of target sequences can be estimated with the Ct method, and addresses the design and test of reliable qPCR barcodes and probes targeting a single species. Finally, several important experimental considerations are highlighted, including the particular concerns of contamination and inhibition in qPCR.

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Chapitres de livres sur le sujet "Real-Time quantitative PCRs"

Sluijter, J. P. G., G. Pasterkamp et D. P. V. de Kleijn. « Quantitative Real-Time PCR ». Dans Cardiovascular Research, 75–83. Boston, MA : Springer US, 2006. http://dx.doi.org/10.1007/0-387-23329-6_4.

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Broll, Hermann. « Quantitative Real-Time PCR ». Dans Molecular Biological and Immunological Techniques and Applications for Food Chemists, 59–83. Hoboken, NJ, USA : John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470637685.ch3.

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Dotti, Isabella, Ermanno Nardon, Danae Pracella et Serena Bonin. « Quantitative Real-Time RT-PCR ». Dans Guidelines for Molecular Analysis in Archive Tissues, 121–32. Berlin, Heidelberg : Springer Berlin Heidelberg, 2011. http://dx.doi.org/10.1007/978-3-642-17890-0_25.

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Martinez, Jeanelle M., et Nigel J. Walker. « Real-Time and Quantitative PCR ». Dans Toxicogenomics, 147–63. Hoboken, NJ, USA : John Wiley & Sons, Inc., 2005. http://dx.doi.org/10.1002/0471669040.ch7.

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Dötsch, Jörg, Ellen Schoof et Wolfgang Rascher. « Quantitative TaqMan Real-Time PCR ». Dans Medical Biomethods Handbook, 305–13. Totowa, NJ : Humana Press, 2005. http://dx.doi.org/10.1385/1-59259-870-6:305.

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Pal, Aruna. « Real-Time or Quantitative PCR ». Dans Springer Protocols Handbooks, 181–209. New York, NY : Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1818-9_9.

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Singh, Charanjeet, et Sinchita Roy-Chowdhuri. « Quantitative Real-Time PCR : Recent Advances ». Dans Clinical Applications of PCR, 161–76. New York, NY : Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3360-0_15.

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Honda, Junichi, et Kotaro Oizumi. « Quantitative Analysis of CMV in Infected Mice on the LightCycler System ». Dans Rapid Cycle Real-Time PCR, 349–57. Berlin, Heidelberg : Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/978-3-642-59524-0_37.

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Krüger, Petra, Albrecht Wiedenmann, Despina Tougianidou et Konrad Botzenhart. « Quantitative Detection of Cryptosporidium parvum after In Vitro Excystation by LightCycler PCR ». Dans Rapid Cycle Real-Time PCR, 341–48. Berlin, Heidelberg : Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/978-3-642-59524-0_36.

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Caplin, Brian Erich. « Quantification of Human Papilloma Virus Type 16 Using Quantitative Competitive PCR on the LightCycler ». Dans Rapid Cycle Real-Time PCR, 57–64. Berlin, Heidelberg : Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/978-3-642-59524-0_6.

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Actes de conférences sur le sujet "Real-Time quantitative PCRs"

Woudenberg, Timothy M., et J. Stevens. « Quantitative PCR by real-time detection ». Dans Photonics West '96, sous la direction de Gerald E. Cohn, Steven A. Soper et C. H. Winston Chen. SPIE, 1996. http://dx.doi.org/10.1117/12.237619.

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« RqPCRAnalysis : Analysis of Quantitative Real-time PCR Data ». Dans International Conference on Bioinformatics Models, Methods and Algorithms. SciTePress - Science and and Technology Publications, 2013. http://dx.doi.org/10.5220/0004312002020211.

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Sayers, Michael B., et Tara M. Dalton. « A Real-Time Continuous Flow Polymerase Chain Reactor for DNA Expression Quantification ». Dans ASME 2007 International Mechanical Engineering Congress and Exposition. ASMEDC, 2007. http://dx.doi.org/10.1115/imece2007-43058.

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Real-time quantitative Polymerase Chain Reaction (PCR) is an extremely sensitive and reliable method for quantifying gene expression, allowing subtle shifts in gene expression to be easily monitored. Currently, stationary real-time PCR is readily achieved using fluorescent labels which increase in fluorescence as the DNA is exponentially amplified. Quantitative PCR is used in a myriad of applications. However currently most commercial real-time PCR devices are batch process stationary well based systems, limiting their throughput. Continuous flow microfluidic PCR devices have allowed for advancement in terms of improved PCR throughput and reduced reagent usage. As part of an overall total analysis system a device integrating all the functional steps of continuous flow realtime quantitative PCR has been designed and fabricated. Initially the PCR reaction mixture is segmented into nano-litre PCR reactors which are then thermally cycled on a two temperature fifty cycle flow-through PCR device, which allows laser induced fluorescent imaging of the nanoreactors. Previous studies into continuous flow PCR have demonstrated endpoint fluorescent measurements, however this research allows PCR nanoreactors to be fluorescently monitored after every PCR thermal cycle. Fluorescent optical monitoring is achieved through laser excitation of the nanoreactors while a Charged Coupled Device (CCD) camera is used to record the fluorescent emissions from the nanoreactors. Intensity analysis of the recorded images is then preformed using MATLAB to accurately determine the fluorescence intensity level, thereby allowing real-time quantitative amplification curves to be generated. This has major advantages over existing continuous flow PCR devices which use endpoint fluorescence and capillary electrophoresis, as the amplification curves allow far more information to be gleaned and allow the initial DNA template concentration to be accurately determined.

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Ma, Yutong, Liang Zeng et Jianhuan Zhang. « A fluorescence detection optical system for real-time quantitative PCR ». Dans Optical Design and Testing X, sous la direction de Rengmao Wu, Osamu Matoba, Yongtian Wang et Tina E. Kidger. SPIE, 2020. http://dx.doi.org/10.1117/12.2574901.

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« Quantitative real-time PCR as a supplementary tool for molecular cytogenetics ». Dans Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 2019. http://dx.doi.org/10.18699/plantgen2019-044.

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« Detection of Gene Expression from Vitis by Real Time Quantitative RT-PCR ». Dans 2018 4th World Conference on Control, Electronics and Computer Engineering. Francis Academic Press, 2018. http://dx.doi.org/10.25236/wccece.2018.15.

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Huai, Yahong, et Shangzhong Xu. « Real-time Fluorescence Quantitative PCR and Prediction of Bovine Trf 2 Protein Funtion ». Dans International Conference on Biomedical and Biological Engineering. Paris, France : Atlantis Press, 2016. http://dx.doi.org/10.2991/bbe-16.2016.30.

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Daly, John, et Mark Davies. « A Quantitative Free Convection DNA Amplifier ». Dans ASME/JSME 2007 Thermal Engineering Heat Transfer Summer Conference collocated with the ASME 2007 InterPACK Conference. ASMEDC, 2007. http://dx.doi.org/10.1115/ht2007-32381.

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The Polymerase Chain Reaction (PCR) has been used extensively to amplify targeted nucleic acids for many applications in molecular biology and, increasingly, in medical diagnostics. Outlined in this paper is a PCR device which takes account of the advantages offered by free convection. The design is, in it fundamental format a time-wise isothermal well-based thermocycler. A temperature gradient induced across the well causes convection forces to circulate the sample through the required temperatures necessary for amplification. Quantitative amplification is demonstrated with real time measurements of SYBR Green I fluorescence within the free convective DNA amplifier. Amplification of an 86-bp fragment of the pGEM®-T vector (Promega) is performed in a 25μl volume in eight minutes. A 10-fold dilution series and methods for calculating effective cycle times are presented. Also detailed within this paper are PIV and thermal imaging results of the free convection cavity. This device presents an opportunity for the development of a practical and inexpensive gene-expression measurement system.

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Taha, Aza, Katan Ali et Furat Sabeer. « Quantitation assay of hepatitis C virus RNA using real-time PCR technique ». Dans 4th International Scientific Conference of Cihan University-Erbil on Biological Sciences. Cihan University-Erbil, 2017. http://dx.doi.org/10.24086/bios17.22.

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Botteldoorn, N., H. Werbrouck, N. Rijpens, E. van Coillie, M. Heyndrickx et L. Herman. « Expression study by real-time quantitative RT-PCR of the Salmonella typhimurium mntH gene ». Dans Second International Symposium on Epidemiology and Control of Salmonella in Pork. Iowa State University, Digital Press, 2003. http://dx.doi.org/10.31274/safepork-180809-463.

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Rapports d'organisations sur le sujet "Real-Time quantitative PCRs"

Harris, D. L. Hank, Isabel Turney Harris, James S. Dickson, Stephen Gaul, Brad T. Bosworth et Lori Feldmann. Quantitative Real-time PCR (qPCR) for the Determination of Salmonella Levels in Lairage. Ames (Iowa) : Iowa State University, janvier 2009. http://dx.doi.org/10.31274/ans_air-180814-1009.

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Sykes, Mark, Rati Bell, Joseph Holland, Kate Perkins et Joy Kaye. Inter-laboratory collaborative trial of real-time PCR method for the relative quantitation of horse DNA and pork DNA in raw and processed beef DNA phases 1 and 2. Food Standards Agency, avril 2023. http://dx.doi.org/10.46756/sci.fsa.qbu570.

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The project describes the full international interlaboratory collaborative trial to define the performance characteristics of the real-time PCR method for horse and pork DNA in raw and processed beef matrix.

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Zhelev, Doncho V., Christopher Dupuis, Suelynn Ren, Anna Le, Mia Hunt et Henry Gibbons. Single Nucleotide Polymorphisms (SNP)-specific Quantitative Real Time Polymerase Chain Reaction (PCR) Assay for Analyzing Competition and Emergence of the Military Hypersporulating Strains of Bacillus Atrophaeous var. Globigii. Fort Belvoir, VA : Defense Technical Information Center, septembre 2012. http://dx.doi.org/10.21236/ada570597.

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Seroussi, Eyal, et George Liu. Genome-Wide Association Study of Copy Number Variation and QTL for Economic Traits in Holstein Cattle. United States Department of Agriculture, septembre 2010. http://dx.doi.org/10.32747/2010.7593397.bard.

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Copy number variation (CNV) has been recently identified in human and other mammalian genomes and increasing awareness that CNV might be a major source for heritable variation in complex traits has emerged. Despite this, little has been published on CNVs in Holsteins. In order to fill this knowledge-gap, we proposed a genome-wide association study between quantitative trait loci (QTL) for economic traits and CNV in the Holstein cattle. The approved feasibility study was aimed at the genome-wide characterization of CNVs in Holstein cattle and at the demonstrating of their possible association with economic traits by performing the activities of preparation of DNA samples, Comparative Genomic Hybridization (CGH), initial association study between CNVs and production traits and characterization of CNVSNP associations. For both countries, 40 genomic DNA samples of bulls representing the extreme sub-populations for economically important traits were CGH analyzed using the same reference genome on a NimbleGen tiling array. We designed this array based on the latest build of the bovine genome (UMD3) with average probe spacing of 1150 bases (total number of probes was 2,166,672). Two CNV gene clusters, PLA2G2D on BTA2 and KIAA1683 on BTA7 revealed significant association with milk percentage and cow fertility, respectively, and were chosen for further characterization and verification in a larger sample using other methodologies including sequencing, tag SNPs and real time PCR (qPCR). Comparison between these four methods indicated that there is under estimation of the number of CNV loci in Holstein cattle and their complexity. The variation in sequence between different copies seemed to affect their functionality and thus the hybridization based methods were less informative than the methods that are based on sequencing. We thus conclude that large scale sequencing effort complemented by array CGH should be considered to better detect and characterize CNVs in order to effectively employ them in marker-assisted selection.

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Ghanim, Murad, Joe Cicero, Judith K. Brown et Henryk Czosnek. Dissection of Whitefly-geminivirus Interactions at the Transcriptomic, Proteomic and Cellular Levels. United States Department of Agriculture, février 2010. http://dx.doi.org/10.32747/2010.7592654.bard.

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Our project focuses on gene expression and proteomics of the whitefly Bemisia tabaci (Gennadius) species complex in relation to the internal anatomy and localization of expressed genes and virions in the whitefly vector, which poses a major constraint to vegetable and fiber production in Israel and the USA. While many biological parameters are known for begomovirus transmission, nothing is known about vector proteins involved in the specific interactions between begomoviruses and their whitefly vectors. Identifying such proteins is expected to lead to the design of novel control methods that interfere with whitefly-mediated begomovirus transmission. The project objectives were to: 1) Perform gene expression analyses using microarrays to study the response of whiteflies (B, Q and A biotypes) to the acquisition of begomoviruses (Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV). 2) Construct a whitefly proteome from whole whiteflies and dissected organs after begomovirus acquisition. 3) Validate gene expression by q-RTPCR and sub-cellular localization of candidate ESTs identified in microarray and proteomic analyses. 4) Verify functionality of candidate ESTs using an RNAi approach, and to link these datasets to overall functional whitefly anatomical studies. During the first and second years biological experiments with TYLCV and SLCV acquisition and transmission were completed to verify the suitable parameters for sample collection for microarray experiments. The parameters were generally found to be similar to previously published results by our groups and others. Samples from whole whiteflies and midguts of the B, A and Q biotypes that acquired TYLCV and SLCV were collected in both the US and Israel and hybridized to B. tabaci microarray. The data we analyzed, candidate genes that respond to both viruses in the three tested biotypes were identified and their expression that included quantitative real-time PCR and co-localization was verified for HSP70 by the Israeli group. In addition, experiments were undertaken to employ in situ hybridization to localize several candidate genes (in progress) using an oligonucleotide probe to the primary endosymbiont as a positive control. A proteome and corresponding transcriptome to enable more effective protein identification of adult whiteflies was constructed by the US group. Further validation of the transmission route of begomoviruses, mainly SLCV and the involvement of the digestive and salivary systems was investigated (Cicero and Brown). Due to time and budget constraints the RNAi-mediated silencing objective to verify gene function was not accomplished as anticipated. HSP70, a strong candidate protein that showed over-expression after TYLCV and SLCV acquisition and retention by B. tabaci, and co-localization with TYLCV in the midgut, was further studies. Besides this protein, our joint research resulted in the identification of many intriguing candidate genes and proteins that will be followed up by additional experiments during our future research. To identify these proteins it was necessary to increase the number and breadth of whitefly ESTs substantially and so whitefly cDNAs from various libraries made during the project were sequenced (Sanger, 454). As a result, the proteome annotation (ID) was far more successful than in the initial attempt to identify proteins using Uniprot or translated insect ESTs from public databases. The extent of homology shared by insects in different orders was surprisingly low, underscoring the imperative need for genome and transcriptome sequencing of homopteran insects. Having increased the number of EST from the original usable 5500 generated several years ago to >600,000 (this project+NCBI data mining), we have identified about one fifth of the whitefly proteome using these new resources. Also we have created a database that links all identified whitefly proteins to the PAVEdb-ESTs in the database, resulting in a useful dataset to which additional ESTS will be added. We are optimistic about the prospect of linking the proteome ID results to the transcriptome database to enable our own and other labs the opportunity to functionally annotate not only genes and proteins involved in our area of interest (whitefly mediated transmission) but for the plethora of other functionalities that will emerge from mining and functionally annotating other key genes and gene families in whitefly metabolism, development, among others. This joint grant has resulted in the identification of numerous candidate proteins involved in begomovirus transmission by B. tabaci. A next major step will be to capitalize on validated genes/proteins to develop approaches to interfere with the virus transmission.

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