Verification of quantification standards used in quantitative PCR by droplet digital PCR

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F18%3AN0000149" target="_blank" >RIV/00027162:_____/18:N0000149 - isvavai.cz</a>

Result on the web

<a href="http://www.eavld2018.org/images/files/EAVLD_2018-Abstract_book.pdf" target="_blank" >http://www.eavld2018.org/images/files/EAVLD_2018-Abstract_book.pdf</a>

DOI - Digital Object Identifier

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Result language

angličtina

Original language name

Verification of quantification standards used in quantitative PCR by droplet digital PCR

Original language description

5th Congress of the European Association of Veterinary Laboratory Diagnostics, MCE BUSINESS Conference Centre, Brussels, Belgium, 14. – 17.10.2018 – presentation. Introduction. Currently, quantitative PCR (qPCR) is one of the most widely used molecular biological methods for the detection of microbial pathogens. Moreover, qPCR allows to quantify microbial load according to the standard calibration curve. Therefore, precise determination of the DNA quantity in the standard is essential. Spectrophotometry is used most often for this purpose, but the spectrophotometric measurement of the nucleic acid absorbance is affected by its purity, which may lead to incorrect dilution of the standard. A promising tool for accurate quantification of these standards is the recently developed droplet digital PCR (ddPCR) method, which provides absolute quantification without the need for a calibration curve. The aim of this study was to assess suitability of ddPCR for the independent verification of qPCR quantification standards. Material and methods. We have selected 4 qPCR assays for the quantification of microbial pathogens and analyzed linearized and circular form of plasmids by qPCR and ddPCR. The effect of repeated plasmid isolation and usage of different plasmid purification kits was assessed as well. Results. Spectrophotometric measurement of the absorbance of the nucleic acid overestimated by 30-50%. The results show that the linearized plasmid was more suitable for the standard than the circular, since amplification of the target nucleic acid sequence is better accessible. Discussion and Conclusion. Using ddPCR, we have verified the four selected quantification standards used in our laboratory. Due to the variability in plasmid batch-to-batch preparation, ddPCR can provide a very powerful tool for the control of plasmid production in time.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10606 - Microbiology

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

2018

Confidentiality

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