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

Kód výsledku v 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>

Výsledek na webu

<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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Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10606 - Microbiology

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

2018

Kód důvěrnosti údajů

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