Quantification of qPCR standards using digital PCR

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F19%3AN0000051" target="_blank" >RIV/00027162:_____/19:N0000051 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.med.muni.cz/tomdny/Program_2019.pdf" target="_blank" >https://www.med.muni.cz/tomdny/Program_2019.pdf</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Quantification of qPCR standards using digital PCR

Popis výsledku v původním jazyce

XXVIII. Tomasek days of young microbiologists, 6th and 7th June 2019 at Brno – lecture. Quantitative real-time PCR (qPCR) is nowadays one of the most commonly used molecular biological methods for the detection and quantification of pathogenic microorganisms. To quantify pathogens, it is necessary to construct a calibration curve from quantification standards containing known amounts of plasmids, genomic DNA, or other nucleic acid molecules. Therefore, determining the exact amount of nucleic acid copies in the standard is essential for the correct quantification of pathogens in the sample. Nucleic acid absorbance measurement is most commonly used for this purpose, however, insufficient purity can affect the results of measurement and consequently dilution quantification standards. Digital PCR, which allows absolute quantification of the target nucleic acid, appears to be a promising tool for independent verification of quantification standards, as it is not dependent on the construction of a calibration curve. The principle of digital PCR is a division of the initial sample to several thousands of reaction compartments in which amplification of nucleic acid takes place separately. The reaction compartments may be emulsion droplets (droplet digital PCR) or wells located on a small digital microchip (chip digital PCR). After amplification, the concentration of target DNA is calculated from the number of positive reaction compartments using Poisson statistics. The aim of this work was to determine and compare concentrations of 45 quantification standards diluted according to absorbance using digital PCR and to determine the effect of using different isolation kits on the preparation of quantification standards.

Název v anglickém jazyce

Quantification of qPCR standards using digital PCR

Popis výsledku anglicky

XXVIII. Tomasek days of young microbiologists, 6th and 7th June 2019 at Brno – lecture. Quantitative real-time PCR (qPCR) is nowadays one of the most commonly used molecular biological methods for the detection and quantification of pathogenic microorganisms. To quantify pathogens, it is necessary to construct a calibration curve from quantification standards containing known amounts of plasmids, genomic DNA, or other nucleic acid molecules. Therefore, determining the exact amount of nucleic acid copies in the standard is essential for the correct quantification of pathogens in the sample. Nucleic acid absorbance measurement is most commonly used for this purpose, however, insufficient purity can affect the results of measurement and consequently dilution quantification standards. Digital PCR, which allows absolute quantification of the target nucleic acid, appears to be a promising tool for independent verification of quantification standards, as it is not dependent on the construction of a calibration curve. The principle of digital PCR is a division of the initial sample to several thousands of reaction compartments in which amplification of nucleic acid takes place separately. The reaction compartments may be emulsion droplets (droplet digital PCR) or wells located on a small digital microchip (chip digital PCR). After amplification, the concentration of target DNA is calculated from the number of positive reaction compartments using Poisson statistics. The aim of this work was to determine and compare concentrations of 45 quantification standards diluted according to absorbance using digital PCR and to determine the effect of using different isolation kits on the preparation of quantification standards.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/QK1810212" target="_blank" >QK1810212: Rychlé, komplexní a multiplexní metody pro simultánní detekci původců alimentárních onemocnění v potravinách živočišného a rostlinného původu</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

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