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

Martina Beinhauerova,Maria Beatrice Boniotti,Barbara Bertasi,Vladimir Babak,Petr Kralik

doi:10.3389/fmolb.2020.00155

Abstract

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.

Highlights

Quantitative PCR is currently the method of choice for nucleic acid detection and quantification of various microbial pathogens due to its high sensitivity, specificity, reproducibility, and wide dynamic range
Its theoretical limit of detection was set at three copies per Quantitative PCR (qPCR) reaction, assuming its ability to detect a single copy of the target nucleic acid (Bustin et al, 2009). qPCR is used in various applications in a wide range of areas including food safety and healthcare digital PCR (dPCR) for Quantification Standards Verification (Kralik et al, 2011; Alidjinou et al, 2017; Walker et al, 2017)
In order to determine the absolute number of microbial pathogens in a sample using qPCR, it is necessary to create a calibration curve derived from a serially diluted quantification standard containing a known amount of copies or concentration of plasmids, genomic DNA or other nucleic acid molecules carrying target genes

Summary

Introduction

Quantitative PCR (qPCR) is currently the method of choice for nucleic acid detection and quantification of various microbial pathogens due to its high sensitivity, specificity, reproducibility, and wide dynamic range. In order to determine the absolute number of microbial pathogens in a sample using qPCR, it is necessary to create a calibration curve derived from a serially diluted quantification standard containing a known amount of copies or concentration of plasmids, genomic DNA or other nucleic acid molecules carrying target genes. The concentration of nucleic acid of a standard is determined based on spectrophotometric or fluorometric measurements of nucleic acid absorbance or fluorescence, respectively (Kline et al, 2009), subsequently the number of DNA copies can be calculated according to the molecular weight of DNA material. Since quantification standards are not uniform among laboratories performing quantification of pathogenic microorganisms using qPCR, the determination of the number of pathogens in various laboratories using different qPCR standards is not comparable, which is reflected in differing interpretations of results (Pavsic et al, 2015)

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