Detection of coeliac disease predisposition using dna biosensor arrays

Coeliac disease (CD) is a small intestinal inflammation, affecting genetically susceptible individuals, triggered by ingestion of certain cereals. Prevalence of CD in Europe and US is about 1%. Most of CD cases remain undiagnosed for many years because of highly variable clinical spectrum and atypical presentation. The gold standard test for CD diagnosis is the small-intestinal biopsy, an invasive and expensive test. Strong relation between CD and Human Leukocyte Antigens (HLA) has been proved, as 95% of the CD patients carrying HLA DQ2 heterodimer and 5% carrying DQ8 heterodimer. DQ2 and DQ8 negative individuals have been shown to be very unlikely to develop CD. The overall objective of the thesis is to develop a rapid, ease to use, cost effective diagnostic genosensor array for DQ2/DQ8 typing as a diagnostic tool for CD predisposition. Two different methods have been investigated in parallel. In the first method, the genosensor array was developed employing the SSOP method, by designing different allele specific probes. Whilst in the second approach, the SSP technique exploiting a novel approach for the direct and rapid detection of a double stranded PCR amplicon was investigated. In both approaches, assay conditions were optimised and finally analysis of real clinical samples was performed.La enfermedad celíaca (EC) es una inflamación del intestino delgado, que afecta a individuos genéticamente susceptibles, provocada por la ingestión de algunos cereales. La prevalencia de EC en Europa y los EE.UU. es de aproximadamente 1%. La mayoría de los casos de CD sin diagnosticar durante muchos años porque de espectro clínico es muy variable y presentación atípica. La prueba estándar de oro para el diagnóstico de EC es la biopsia del intestino delgado, una prueba invasiva y costosa. Fuerte relación entre la CD y antígenos leucocitarios humanos (HLA) se ha demostrado, con un 95% de los pacientes con EC portadores del antígeno HLA DQ2 heterodímero y el 5% llevan DQ8 heterodímero. Personas negativos DQ2 y DQ8 han demostrado ser muy poco probable que desarrollen CD. El objetivo general de la tesis es el desarrollo de una manera rápida, fácil de utilizar, rentable matriz genosensor de diagnóstico para DQ2/DQ8 escribir como una herramienta de diagnóstico para la predisposición de CD. Dos métodos diferentes han sido investigados en paralelo. En el primer método, la matriz genosensor se desarrolló empleando el método SSOP, mediante el diseño de sondas diferentes alelos específicos. Mientras que en el segundo enfoque, la técnica SSP explotar un nuevo enfoque para la detección directa y rápida de una doble cadena de amplificación de PCR se investigó. En ambos métodos, condiciones de ensayo fueron optimizadas y finalmente el análisis de muestras clínicas reales se realizó

Design of Gaussia luciferase-based bioluminescent stem-loop probe for sensitive detection of HIV-1 nucleic acids

Here we describe the design of a bioluminescent stem-loop probe for the sensitive detection of HIV-1 spliced RNA. In this study, we employed Gaussia luciferase (GLuc), a bioluminescent protein that has several advantages over other bioluminescent proteins, including smaller size, higher bioluminescent intensity, and chemical and thermal stability. GLuc was chemically conjugated to the DABCYL-modified stem-loop probe (SLP) and was purified with a 2-step process to remove unconjugated GLuc and SLP. The binding of the target RNA to the loop region of the SLP results in the open conformation separating the stem part of SLP. GLuc conjugated to the stem acts as a reporter that produces light by a chemical reaction upon adding its substrate, coelenterazine in the presence of the target, while DABCYL serves as a quencher of bioluminescence in the closed conformation of SLP in the absence of the target. The optimized GLuc based-SLP assay resulted in a signal-to-background ratio of 47, which is the highest reported with bioluminescent SLPs and is significantly higher compared to traditional fluorescence-based SLPs that yield low signal to background ratio. Moreover, the assay showed an excellent selectivity against a single and double mismatched nucleic acid target, low detection limit, and ability to detect spiked HIV-1 RNA in human serum matrix

Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing

Coeliac disease is an inflammation of the small intestine triggered by gluten ingestion. We present a fluorescent genosensor, exploiting molecular-beacon-functionalized gold nanoparticles, for the identification of human leukocyte antigen (HLA) DQ2 gene, a key genetic factor in coeliac disease. Optimization of sensor performance was achieved by tuning the composition of the oligonucleotide monolayer immobilized on the gold nanoparticle and the molecular beacon design. Co-immobilization of the molecular beacon with a spacing oligonucleotide (thiolated ten-thymine oligonucleotide) in the presence of ten-adenine oligonucleotides resulted in a significant increase of the sensor response owing to improved spacing of the molecular beacons and extension of the distance from the nanoparticle surface, which renders them more available for recognition. Further increase in the response (approximately 40%) was shown to be achievable when the recognition sequence of the molecular beacon was incorporated in the stem. Improvement of the specificity of the molecular beacons was also achieved by the incorporation within their recognition sequence of a one-base mismatch. Finally, gold nanoparticles functionalized with two molecular beacons targeting the DQA1*05* and DQB1*02* alleles allowed the low-resolution typing of the DQ2 gene at the nanomolar level

Strengthening of posture, for the eldest preschool-children physical activity during lessons

Bakalaura darba tēma. Vecākā pirmsskolas vecuma bērnu stājas nostiprināšana sporta nodarbībās. Bakalaura darba autors. Anda Priedoliņa. Bakalaura darba mērķis. Teorētiski analizēt un praktiski pētīt vecākā pirmsskolas vecuma bērnu stājas sekmēšanas iespējas sporta nodarbībās. Bakalaura darba uzdevumi: 1.Analizēt pedagoģijas un psiholoģijas zinātnieku atziņas par stāju un vecākā pirmsskolas vecuma bērnu attīstību. 2.Izzināt sporta nodarbību struktūru, saturu un stājas nostiprināšanas iespējas tajās. 3.Pētīt stājas sekmēšanas iespējas sporta nodarbībās vecākā pirmsskolas vecuma bērniem. Bakalaura darba saturs. Darbs sastāv no 2 daļām –teorētiskās un empīriskās. Teorētiskā daļā tika analizēta literatūra par stāju un tas veidiem, stājas ietekmi uz bērna veselību un attīstību, 6-7 gadu vecu bērnu attīstības raksturojums, sporta nodarbības nozīme stājas sekmēšanā, fizisko aktivitāšu analizēšana, ar kuru palīdzību var nostiprināt stāju. Empīriskajā daļā tika novērota 10 vecākā pirmsskolas vecuma grupas bērnu stāja un izstrādāti vingrinājuma kompleksi, kas uzlabotu un novērstu novērotos stājas traucējumus. Veiktas pārrunas ar bērniem par viņu vēlamajām aktivitātēm sporta nodarbībā, lai nodarbības padarītu interesantākas un saistošākas bērniem. Vecāku anketēšana par bērnu nodarbošanos un fiziskām aktivitātēm pēc pirmsskolas izglītības iestādes apmeklēšanas, lai noskaidrotu iespējamās problēmas, kas varētu atspoguļoties uz stājas steriotipa veidošanos. Pētījumā secināts, ka vecākā pirmskolas vecuma bērnu stājas nostiprināšana sporta nodarbībā būs veiksmīga, ja pedagogs novērtēs katra skolēna stāju un izveidos nepieciešamo vingrojumu kompleksu novērotajiem stājas traucējumiem, ņemot vērā bērna attīstības līmeni. Lai novērstu sājas traucējumus, ir nepieciešama iesaistīšanās arī no pirmsskolas izglītības iestādes personāla un bērnu vecākiem, kas sniegtu priekšstatu par stājas stereotipu veidošanos, jo stājas traucējumu profilakse var būt ilgstošs un sarežģīts process. Bakalaura darba apjoms 51 lpp. (bez pielikuma), iekļauti 12 attēli, 2 tabulas, 8 pielikumi. Darbā izmantoti 48 literatūras avoti, no kuriem 40 latviešu valodā, 8 svešvalodā. Atslēgas vārdi: sporta nodarbība, stājas nostiprināšana, vecākā pirmsskolas vecuma grupa.Bachelor Thesis Topic. Strengthening of posture, for the eldest preschool-children physical activity during lessons. Bachelor Thesis Author. Anda Priedoliņa. Bachelor Thesis Objective. Theoretically analyze and practically research the possibility to strengthen the posture of the oldest preschool children physical activity during lessons. Bachelor Thesis Tasks: 1.Analyze teaching and psychology scientist conclusions about posture and the oldest preschool children development. 2.Explore the structure and content of Physical Education and the possibilities of posture strengthening in them. 3.Study the posture strengthening possibilities during Physical Education for the oldest preschool children. Bachelor Thesis Content. The Thesis consists of 2 parts – the theoretical and empirical. The theoretical part consists of the analysis of literature on posture and its types, the posture’s influence on a child’s health and development, 6-7 year-old child characterization, the meaning of Physical Education for results in posture, and the analysis of physical activities with which posture can be strengthened. In the empirical part the posture of 10 older preschool age group children was observed and a set of exercises was developed, which would improve and eliminate the observed posture complications. Interviews with children have been conducted to understand their preferred activities in the Physical Education class in order to make the classes more interesting and binding for children. Also, parents were being surveyed about children’s occupation and physical activities after attending the preschool to identify the possible problems that may reflect on the development of posture. The study concludes that strengthening the position of children in the oldest preschool age group will be successful if the teacher evaluates every student’s posture and will create the necessary set of exercises for the observed posture complications, taking into consideration the development stage of the child. In order to prevent posture complications, there is also need of involvement from the staff of the preschool, as well as from the parents to give the idea of the development of posture stereotypes, since prevention of posture complications can be a lengthy and difficult process. Bachelor Thesis Volume. 51 pages. (without appendices), included 12 pictures, 2 tables, 8 appendices. 48 literature sources have been used in the Thesis, from which 40 are in Latvian and 8 in a foreign language. Keywords: Physical Education, posture strengthening, oldest preschool age group

Bioluminescent Protein-Inhibitor Pair in the Design of a Molecular Aptamer Beacon Biosensing System

Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ (IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γ and provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems

CEBADORES ESPECIALES PARA LA REACCION EN CADENA DE LA POLIMERASA

The invention relates to the field of molecular biology techniques, in particular to the polymerase chain reaction (PCR). More specifically, the invention relates to a primers specially designed for the PCR and to the direct detection of a target sequence. This is achieved owing to the special design of the primer and, more specifically, owing to the presence of single-strand tails in the amplification product obtained using said primers. These tails allow the capture of the amplification product on a functionalised substrate with capture probes and/or the simple detection thereof with reporter probes

Bleed-to-read disposable microsystems for the genetic and serological analysis of celiac disease markers with amperometric detection

Coeliac disease is an auto-immune disorder induced by ingestion of gluten in genetically predisposed individuals. Its diagnostics is more accurate using a combination of immunologic and genetic tests to detect of high levels of certain auto-antibodies and the presence human leukocyte antigen HLA-DQ2 or HLA-DQ8 genetic markers. In this work, we report the design and testing of automated microsystems combining sample treatment, storage, fluidic transport and detection in a single platform able to carry out genetic or serologic analysis for detection of celiac disease markers. These microsystems share a common footprint and many fluidic features and are thus able to perform a complete assay. The microsystem for the genetic assay extracts and amplifies the DNA prior to detection, while the serology microsystem contains a filter and chamber for the generation and subsequent dilution of plasma. The performance of both platforms is demonstrated and compared with reference methods with an excellent correlation, which makes the developed platform amenable for clinical studies

Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained

Low-medium resolution HLA-DQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay

Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained