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.

Controllo Alimenti Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna Brescia Italy

Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

Department of Food and Feed Safety Veterinary Research Institute Brno Czechia

Department of Hygiene and Technology of Food of Animal Origin and of Gastronomy Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Czechia

Tecnologie Biologiche Applicate Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna Brescia Italy

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Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR