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Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR

Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pat...

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Autores principales: Beinhauerova, Martina, Babak, Vladimir, Bertasi, Barbara, Boniotti, Maria Beatrice, Kralik, Petr
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403525/
https://www.ncbi.nlm.nih.gov/pubmed/32850953
http://dx.doi.org/10.3389/fmolb.2020.00155
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author Beinhauerova, Martina
Babak, Vladimir
Bertasi, Barbara
Boniotti, Maria Beatrice
Kralik, Petr
author_facet Beinhauerova, Martina
Babak, Vladimir
Bertasi, Barbara
Boniotti, Maria Beatrice
Kralik, Petr
author_sort Beinhauerova, Martina
collection PubMed
description Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR.
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spelling pubmed-74035252020-08-25 Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR Beinhauerova, Martina Babak, Vladimir Bertasi, Barbara Boniotti, Maria Beatrice Kralik, Petr Front Mol Biosci Molecular Biosciences Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR. Frontiers Media S.A. 2020-07-29 /pmc/articles/PMC7403525/ /pubmed/32850953 http://dx.doi.org/10.3389/fmolb.2020.00155 Text en Copyright © 2020 Beinhauerova, Babak, Bertasi, Boniotti and Kralik. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Beinhauerova, Martina
Babak, Vladimir
Bertasi, Barbara
Boniotti, Maria Beatrice
Kralik, Petr
Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
title Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
title_full Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
title_fullStr Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
title_full_unstemmed Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
title_short Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
title_sort utilization of digital pcr in quantity verification of plasmid standards used in quantitative pcr
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403525/
https://www.ncbi.nlm.nih.gov/pubmed/32850953
http://dx.doi.org/10.3389/fmolb.2020.00155
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