| Attributes | Values |
|---|
| rdf:type
| |
| rdfs:seeAlso
| |
| Description
| - Hydrolysis probes, also known as TaqMan (Applied Biosystems), are routinely used in qPCR diagnostics. The disadvantage of these probes is that they have to be designed for each target sequence separately, which makes the analysis more expensive. In order to reduce the costs, we can use the UPL probes (Universal ProbeLibrary; Roche), which contain locked nucleic acids and are of short length of just 8-9 nucleotides. The advantage of UPL probes is that they can bind to their targets with increased strength due to the incorporation of locked nucleic acids in their sequence. Their short length allows them to bind to several positions within the whole genome, which could be disadvantageous in some applications. Moreover, the point mutation can lead to the false negative or false positive results due to their short length. This problem can be overcome by using an intercalating dye which has a different fluorescent spectrum than UPL probe and binds unspecifically to any double stranded DNA Thus, we can get two fluorescent signals from one target sequence. Moreover, this intercalating dye can be used in either melting or HRM analysis. We developed the dual labelled two-step RT-qPCR for the detection of selected viruses. The UPL probe #138 was used for Louping ill and Powasan virus and UPL probe #15 was used for Chikungunya virus. The SYTO 61 Red Fluorescent Nucleic Acid Stain (Invitrogen) was added to all reactions as a second fluorescent reporter. The PCR profiles were used according to the UPL probe’s manufacturer instructions. In all pathogens studied (Louping ill virus, Powasan virus and Chikungunya), there were observed no reciprocal overlaps in fluorescent spectra and the melting profiles were clearly distinguished. Our experiments showed that the combination of the UPL probe and SYTO intercalating dye in one qPCR assay represents a highly sensitive, specific, less time-consuming and cheaper method.
- Hydrolysis probes, also known as TaqMan (Applied Biosystems), are routinely used in qPCR diagnostics. The disadvantage of these probes is that they have to be designed for each target sequence separately, which makes the analysis more expensive. In order to reduce the costs, we can use the UPL probes (Universal ProbeLibrary; Roche), which contain locked nucleic acids and are of short length of just 8-9 nucleotides. The advantage of UPL probes is that they can bind to their targets with increased strength due to the incorporation of locked nucleic acids in their sequence. Their short length allows them to bind to several positions within the whole genome, which could be disadvantageous in some applications. Moreover, the point mutation can lead to the false negative or false positive results due to their short length. This problem can be overcome by using an intercalating dye which has a different fluorescent spectrum than UPL probe and binds unspecifically to any double stranded DNA Thus, we can get two fluorescent signals from one target sequence. Moreover, this intercalating dye can be used in either melting or HRM analysis. We developed the dual labelled two-step RT-qPCR for the detection of selected viruses. The UPL probe #138 was used for Louping ill and Powasan virus and UPL probe #15 was used for Chikungunya virus. The SYTO 61 Red Fluorescent Nucleic Acid Stain (Invitrogen) was added to all reactions as a second fluorescent reporter. The PCR profiles were used according to the UPL probe’s manufacturer instructions. In all pathogens studied (Louping ill virus, Powasan virus and Chikungunya), there were observed no reciprocal overlaps in fluorescent spectra and the melting profiles were clearly distinguished. Our experiments showed that the combination of the UPL probe and SYTO intercalating dye in one qPCR assay represents a highly sensitive, specific, less time-consuming and cheaper method. (en)
|
| Title
| - Using TaqMan Assay Followed By Melting Analysis Performed By New-Generation Intercalating Dyes
- Using TaqMan Assay Followed By Melting Analysis Performed By New-Generation Intercalating Dyes (en)
|
| skos:prefLabel
| - Using TaqMan Assay Followed By Melting Analysis Performed By New-Generation Intercalating Dyes
- Using TaqMan Assay Followed By Melting Analysis Performed By New-Generation Intercalating Dyes (en)
|
| skos:notation
| - RIV/70565813:_____/13:#0000290!RIV14-MV0-70565813
|
| http://linked.open...avai/predkladatel
| |
| http://linked.open...avai/riv/aktivita
| |
| http://linked.open...avai/riv/aktivity
| |
| http://linked.open...vai/riv/dodaniDat
| |
| http://linked.open...aciTvurceVysledku
| |
| http://linked.open.../riv/druhVysledku
| |
| http://linked.open...iv/duvernostUdaju
| |
| http://linked.open...titaPredkladatele
| |
| http://linked.open...dnocenehoVysledku
| |
| http://linked.open...ai/riv/idVysledku
| - RIV/70565813:_____/13:#0000290
|
| http://linked.open...riv/jazykVysledku
| |
| http://linked.open.../riv/klicovaSlova
| - Universal ProbeLibrary probes; qPCR; pathogen detection (en)
|
| http://linked.open.../riv/klicoveSlovo
| |
| http://linked.open...ontrolniKodProRIV
| |
| http://linked.open...v/mistoKonaniAkce
| - TUM, Weihenstephaner Berg 3, 85354 Freising
|
| http://linked.open...i/riv/mistoVydani
| - TUM, Freising-Weihenstephan, Germany
|
| http://linked.open...i/riv/nazevZdroje
| - qPCR 2013 Symposium Proceedings
|
| http://linked.open...in/vavai/riv/obor
| |
| http://linked.open...ichTvurcuVysledku
| |
| http://linked.open...cetTvurcuVysledku
| |
| http://linked.open...vavai/riv/projekt
| |
| http://linked.open...UplatneniVysledku
| |
| http://linked.open...iv/tvurceVysledku
| - Procházková, Jiřina
- Bílek, Karel
- Kubíček, Oldřich
|
| http://linked.open...vavai/riv/typAkce
| |
| http://linked.open.../riv/zahajeniAkce
| |
| number of pages
| |
| http://purl.org/ne...btex#hasPublisher
| - TUM, Freising-Weihenstephan, Germany
|
| https://schema.org/isbn
| |
![[RDF Data]](/fct/images/sw-rdf-blue.png)
![[cxml]](/fct/images/cxml_doc.png)
![[csv]](/fct/images/csv_doc.png)
![[text]](/fct/images/ntriples_doc.png)
![[turtle]](/fct/images/n3turtle_doc.png)
![[ld+json]](/fct/images/jsonld_doc.png)
![[rdf+json]](/fct/images/json_doc.png)
![[rdf+xml]](/fct/images/xml_doc.png)
![[atom+xml]](/fct/images/atom_doc.png)
![[html]](/fct/images/html_doc.png)