Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification

A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection

Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field

Molecular techniques for pathogen identification and fungus detection in the environment

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here

Genetic Analyses using Rolling Circle or PCR Amplified Padlock Probes

Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis. The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization. Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format

Dero Königl: Mayt: und Reiche Schweden ... General Feldtmarschall Johan Baner uff Mühlhammer ... Fügen allen und Jeden ... zuwissen/ welcher gestalt Johann Stalman/ so sich nennet den Eltern/ hiebevorn in diesen beyden Ertz: und Stifftern Magdeburg und Halberstadt verordneter Cantzler und Königl. Schwedischer KriegsRath/ und Jacob Kappaun ... beyde Königl. Ministri ... zu declariren/ zu verfolgen und zu bestraffen ... : Geschehen Magdeburg am 23. Aprilis Anno 1635. ...

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The role of web design for trust in e-commerce : How different design aspects influence consumers trust for e-commerce websites

E-commerce is a phenomenon that constantly evolves and expands which demands high standards towards web design to enhance the functionality and usability of websites. For this bachelor thesis, semi-structured interviews were applied to collect data on how several web design aspects impact e-commerce users’ trust towards e-commerce websites. The use of a qualitative method was conducted due to its efficiency in the collection of detailed information regarding e-commerce consumers’ opinions and thoughts about web design and its impact on their trust. Eight participants of varying ages were interviewed, which in a distinct way manifest that design aspects in many ways are crucial for the influence of users’ trust on e-commerce websites. This bachelor thesis indicates that web design impacts consumers trust, and that certain aspects of a website’s design are more powerful to either enhance or lower trust. The use of functional navigation design can be argued to be one of the more powerful design aspects for the enhancement of trust towards website’s. Navigation design constitutes a large part of a websites pragmatic qualities which has been found to be essential for the trustworthiness of websites

HIV-positiva patienters upplevelser av stigmatisering i vården

 Human immunodeficiency virus (HIV) är ett retrovirus vilket går till angrepp på immunförsvaret och orsakar “immunbrist” hos människan. Viruset kan spridas mellan människor via oskyddat samlag, blodtransfusioner, organtransplantationer eller vid användning av kontaminerade sprutor. I begynnelsen av virusets spridning ansågs homosexuella män vara orsaken till utbrottet, vilket innebar att stigmatisering mot människor med HIV-diagnos tog sin början. HIV är en infektionssjukdom som trots vår moderna tidsepok fortfarande omgärdas av fördomar, rädslor och okunskap. Syftet med denna deskriptiva litteraturstudie var att beskriva HIV-positiva patienters upplevelser av stigmatisering i vården.  Litteraturstudien var baserad på 13 vetenskapliga artiklar som sökts fram i databasen Cinahl och Medline via PubMed. Studierna i denna litteraturstudie påvisade att stigmatisering upplevdes bland HIV-positiva patienter i deras möte med vården. Samtidigt pekade en del av resultatet även på goda erfarenheter där stigmatisering inte varit uttalad. I den del där stigmatisering varit påtaglig beskrevs det främst i form av bemötandeproblem, bristande attityd eller sekretessbrott mot patienterna. Vidare visade resultatet att social stigmatisering hade stor inverkan på individens självkänsla och påverkade således också patientens uppfattning kring sjukdomen. Resultatet i denna litteraturstudie visar att patienter med sjukdomen HIV möter stigmatisering och diskriminering från vårdpersonal när de söker vård. Detta har främst visat sig bero på okunskap kring sjukdomen men även en rädsla för att bli smittad, då kunskapen om hur den smittar ofta är problemet. I arbetet med att säkerställa vårdens kvalitet krävs preventiva åtgärder gällande stigmatisering. Därför kan det också antas att kunskap och utbildning är nyckeln till framgång i detta ämne.

Transport and logistics optimization for Delsbo Candle AB

Tredjepartslogistik i Sverige är en allt växande marknad med många aktörer. För företag som vill lägga ut delar av sin logistisk på dessa aktörer är valet av aktör inte alltid självklart. Ett företag som står inför detta val är stearinljustillverkaren Delsbo Candle AB. Detta arbete tar avstamp i Delsbo Candle existerande transportstruktur och presenterar en globalt optimal lösning, ur ett kostnadsperspektiv, utifrån företagets förutsättningar och begränsningar. Den globalt optimala lösningen utgörs av tre tredjepartslogistik aktörer belägna i Stockholm, Norrköping och Jönköping med en minimal transport- och hanteringskostnad på 45 986 kr per månad. Vidare presenteras en analys av samverkan mellan Delsbo Candle organisationsstruktur och de effekter som outsourcing medför ur ett organisatoriskt perspektiv. Analysen visar bland annat att Delsbo Candle riskerar att utsättas för en fördröjd beslutsprocess och förlust av strategiskt viktig kunskap.The third-party logistics market is rapidly growing in Sweden with actors situated all over the country. Nonetheless are companies that are interested in outsourcing parts of their logistic operation not always certain which actors to choose. A company that faces this particular decision is candle manufacturer Delsbo Candle. This project explores Delsbo Candle’s existing organisation structure and it’s conditions and limitations, followed by a presentation of a global optimal solution from a cost-effective perspective. The global optimal solution consists of using three third-party logistics actors situated in Stockholm, Norrköping och Jönköping with a minimal transport and handling cost of 45 986 kr a month. Furthermore the project explores the concurrence between Delsbo Candle’s organisation structure and the effects of outsourcing from an organisational perspective. The analysis shows that there is a risk that Delsbo Candle will be exposed to delay in decision making and loss of strategically important knowled