Use of Universal ProbeLibrary Probes for Detection of Highly Pathogenic Biological Agents

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F70565813%3A_____%2F19%3AN0000049" target="_blank" >RIV/70565813:_____/19:N0000049 - isvavai.cz</a>

Výsledek na webu

—

DOI - Digital Object Identifier

—

Jazyk výsledku

angličtina

Název v původním jazyce

Use of Universal ProbeLibrary Probes for Detection of Highly Pathogenic Biological Agents

Popis výsledku v původním jazyce

In the world of the real-time PCR, there are two approaches how to obtain a fluorescent signal from the tube. Some investigators insist that unspecific intercalating dyes are a sufficient and cost-effective system to obtain a reliable signal. Others feel that only probe-based assays are the right approach. The UPL probes (Universal ProbeLibrary; Roche) offer a compromise that provides the specificity of a hydrolysis probe but does not require a unique probe for each target. The UPL probes use a nucleotide chemistry called LNA (Locked Nucleic Acid), which allows very short (8-9 bases) oligonucleotides to be effective in hybridization with the target. LNA allows the melting temperature of the short probes to be unusually high. Hence, the objective is to demonstrate the use of the UPL probe for the detection of highly pathogenic biological agents. Moreover, we routinely combine the UPL probes with an intercalating dye (SYTO 61 dye; Invitrogen) for the verification of results. The detection strategy based on the use of the UPL probes was validated on various bacteria e.g. Brucella sp., Vibrio cholerae, Salmonella Typhi, Chlamydophila psittaci, further on selected Rickettsiae, as well as in the detection of viruses using RT-qPCR e.g. Chapare, Chikungunya, Ebola, Hantaan, Lujo, Seoul or Sin Nombre virus. Thanks to the shortness of the UPL probes sequences, it is possible to combine one UPL probe with multiple loci (in the combination with different pairs of primers). This feature was used for the detection of both Zaire and Sudan ebolavirus and for the detection of both the Brucella genus and Vibrio cholerae by the probes no. 138 and no. 11, respectively. All the presented assays were validated according to the MIQE guidelines. In summary, the UPL probes can be used for the rapid, flexible and reliable detection and quantification of highly pathogenic biological agents. Furthermore, it is able to easily design gene-specific quantification assays using the UPL probes and the free web-based ProbeFinder software at the Assay Design Center.

Název v anglickém jazyce

Use of Universal ProbeLibrary Probes for Detection of Highly Pathogenic Biological Agents

Popis výsledku anglicky

In the world of the real-time PCR, there are two approaches how to obtain a fluorescent signal from the tube. Some investigators insist that unspecific intercalating dyes are a sufficient and cost-effective system to obtain a reliable signal. Others feel that only probe-based assays are the right approach. The UPL probes (Universal ProbeLibrary; Roche) offer a compromise that provides the specificity of a hydrolysis probe but does not require a unique probe for each target. The UPL probes use a nucleotide chemistry called LNA (Locked Nucleic Acid), which allows very short (8-9 bases) oligonucleotides to be effective in hybridization with the target. LNA allows the melting temperature of the short probes to be unusually high. Hence, the objective is to demonstrate the use of the UPL probe for the detection of highly pathogenic biological agents. Moreover, we routinely combine the UPL probes with an intercalating dye (SYTO 61 dye; Invitrogen) for the verification of results. The detection strategy based on the use of the UPL probes was validated on various bacteria e.g. Brucella sp., Vibrio cholerae, Salmonella Typhi, Chlamydophila psittaci, further on selected Rickettsiae, as well as in the detection of viruses using RT-qPCR e.g. Chapare, Chikungunya, Ebola, Hantaan, Lujo, Seoul or Sin Nombre virus. Thanks to the shortness of the UPL probes sequences, it is possible to combine one UPL probe with multiple loci (in the combination with different pairs of primers). This feature was used for the detection of both Zaire and Sudan ebolavirus and for the detection of both the Brucella genus and Vibrio cholerae by the probes no. 138 and no. 11, respectively. All the presented assays were validated according to the MIQE guidelines. In summary, the UPL probes can be used for the rapid, flexible and reliable detection and quantification of highly pathogenic biological agents. Furthermore, it is able to easily design gene-specific quantification assays using the UPL probes and the free web-based ProbeFinder software at the Assay Design Center.

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/VI20172019063" target="_blank" >VI20172019063: Vývoj nových metodik pro detekci biologických agens v oblastech souvisejících s dodržováním Úmluvy o zákazu biologických zbraní</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů