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

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.

Keywords: absolute quantification; digital PCR; qPCR; quantification plasmid standard; quantity verification; real time PCR.

Figure 1

Comparison of circular and linear form of plasmid standards by ddPCR. The circular plasmids are show in columns E04, F04, and G04, the linear ones in columns E03, F03, and G03. H04 and H03 are corresponding negative controls. The line across all columns indicates a threshold line separating positive and negative droplets situated above and below the line, respectively.

Figure 2

Comparison of six different kits for plasmid DNA isolation. The bars represent the calculated MLE of plasmid standard concentration compared to the theoretical concentration (the values are given in percent).

Figure 3

Comparison of quantity value of 45 linear plasmid standards using ddPCR and cdPCR. The bars represent the calculated MLE of plasmid standard concentration compared to the theoretical concentration (the values are given in percent).

Figure 4

Comparison of the concentrations of selected standards diluted based on spectrophotometric estimation (Absorbance) and re-diluted based on value (MLE) determined by ddPCR (ddPCR). The bars represent the calculated MLE of plasmid standard concentration compared to the theoretical concentration (the values are given in percent).

Figure 5

Comparison of the concentrations of linear plasmid standards diluted based on spectrophotometric estimation in the beginning (Absorbance in the beginning), 19 or 14 months thereafter (Absorbance after 19 or 14 months) and re-diluted based on value (MLE) determined by ddPCR (ddPCR). The bars represent the calculated MLE of plasmid standard concentration compared to the theoretical concentration (the values are given in percent).