Maja Sidstedt 1,2,*,Erica L. Romsos3, Ronny Hedell2,4, Carolyn R. Steffen3, Peter M. Vallone3, Peter Rådström1, Johannes Hedman 1,2
1Applied Microbiology, Department of Chemistry, Lund University, PO Box 124, 221 00 Lund, Sweden
2 Swedish National Forensic Centre, 581 94 Linköping, Sweden
3 Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899-8314, United States
4 Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, 41296 Gothenburg, Sweden;
Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning the sample into hundreds or thousands of minute reactions . By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA con- centration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrices such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds [2,3]. In this study, we present how certain inhibitors disturb dPCR quantification and sug- gest solutions to these problems. Furthermore, we use real-time PCR, dPCR and isothermal titration calorimetry as tools to eluci- date the mechanisms underlying the PCR inhibition. The impact of impurities on dPCR quantification was studied using humic acid as a model inhibitor. We show that the inhibitor-tolerance dif- fers greatly for three different DNA polymerases, illustrating the importance of choosing a DNA polymerase-buffer system that is compatible with the samples to be analysed. Various inhibitory- substances from blood were found to disturb the system in different ways. For example, hemoglobin was found to cause quenching of fluorescence and a dramatic decrease of the number of posi- tive reactions, leading to an underestimation of DNA quantity. IgG caused an increased number of late-starters. The system was more susceptible to inhibition by IgG when single-stranded DNA was used as template, compared with double-stranded DNA. By under- standing more about the mechanisms of PCR inhibitors it will be possible to design more optimal PCR chemistries, improving dPCR detection and quantification.
1 M. Baker, Digital PCR hits its stride, Nat. Methods 9 (2012) 541–544.
2 C. Coudray-Meunier, A. Fraisse, S. Martin-Latil, L. Guillier, S. Delannoy, P. Fach,
S. Perelle, A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples, Int. J. Food Microbiol. 201 (2015) 17–26.
3 T. Hoshino, F. Inagaki, Molecular quantification of environmental DNA using microfluidics and digital PCR, Syst. Appl. Microbiol. 35 (2012) 390–395.
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