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

@article{1672162,
   author = {Beinhauerová, Martina and Babák, Vladimír and Bertasi, Barbara and Boniotti, Maria Beatrice and Králík, Petr},
   article_location = {Lausanne},
   article_number = {July 2020},
   doi = {http://dx.doi.org/10.3389/fmolb.2020.00155},
   keywords = {digital PCR; absolute quantification; quantity verification; quantification plasmid standard; qPCR; real time PCR},
   language = {eng},
   issn = {2296-889X},
   journal = {Frontiers in Molecular Biosciences},
   title = {Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR},
   url = {https://doi.org/10.3389/fmolb.2020.00155},
   volume = {7},
   year = {2020}
}

TY  - JOUR
ID  - 1672162
AU  - Beinhauerová, Martina - Babák, Vladimír - Bertasi, Barbara - Boniotti, Maria Beatrice - Králík, Petr
PY  - 2020
TI  - Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR
JF  - Frontiers in Molecular Biosciences
VL  - 7
IS  - July 2020
SP  - 1-13
EP  - 1-13
PB  - Frontiers Media SA
SN  - 2296889X
KW  - digital PCR
KW  - absolute quantification
KW  - quantity verification
KW  - quantification plasmid standard
KW  - qPCR
KW  - real time PCR
UR  - https://doi.org/10.3389/fmolb.2020.00155
L2  - https://doi.org/10.3389/fmolb.2020.00155
N2  - 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.
ER  - 

BEINHAUEROVÁ, Martina, Vladimír BABÁK, Barbara BERTASI, Maria Beatrice BONIOTTI a Petr KRÁLÍK. Utilization of Digital PCR in Quantity Verification of Plasmid Standards Used in Quantitative PCR. \textit{Frontiers in Molecular Biosciences}. Lausanne: Frontiers Media SA, 2020, roč.~7, July 2020, s.~1-13. ISSN~2296-889X. Dostupné z: https://dx.doi.org/10.3389/fmolb.2020.00155.