Quantification of qPCR standards using digital PCR

Result code in 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>

Result on the web

<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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Result language

angličtina

Original language name

Quantification of qPCR standards using digital PCR

Original language description

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.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10608 - Biochemistry and molecular biology

Project

<a href="/en/project/QK1810212" target="_blank" >QK1810212: Rapid, complex and multiplex methods for simultaneous detection of food-borne pathogens in food of animal and plant origin</a><br>

Continuities

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

Publication year

2019

Confidentiality

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