Abstract
The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as “rain” in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.
Highlights
Droplet digital polymerase chain reaction is a relatively new method enabling quantification without external standards. It has already been established in various fields [1], such as quantification of HIV DNA [2], assessing food containing genetically modified organisms [3,4,5,6] or for the detection and quantification of bacterial pathogens in plants [7] or water samples [8]
We showed that quantification with the Bio-Rad Droplet digital polymerase chain reaction (ddPCR) system was sufficient precise, despite suboptimal cluster formation [16]
In the case of the prfA assay, we showed that direct transfer resulted in strongly biased Ct values when using the chemistry required for ddPCR [16]
Summary
Droplet digital polymerase chain reaction (ddPCR) is a relatively new method enabling quantification without external standards. It has already been established in various fields [1], such as quantification of HIV DNA [2], assessing food containing genetically modified organisms [3,4,5,6] or for the detection and quantification of bacterial pathogens in plants [7] or water samples [8]. The principle of ddPCR is based on the distribution of DNA according to Poisson distribution In this respect, and before amplification, the reaction is divided into many small reactions (20,000 droplets in case of the Bio-Rad System) which either contain DNA or not. Knowing the number of positive/negative droplets and the Poisson distribution, the initial DNA concentration can be PLOS ONE | DOI:10.1371/journal.pone.0168179 December 19, 2016
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