Molecular quantification of bacterial DNA of the positive control (S. pneumoniae ATCC 49619)

For quantification of a positive control (S. pneumoniae ATCC 49619) we used the Quant-IT PicoGreen dsDNA assay kit (Life Technologies, Bleiswijk, The Netherlands). The fluorescence was measured using a LightCycler® 480II real-time PCR analyser (Roche, Almere, The Netherlands). To convert from the concentration in ng/µL to the number of genome copies per µL the genome size, approximately 2.1 million base pairs was estimated [30].

As a comparison for the Picogreen quantification method, quantification of S. pneumoniae ATCC 49619 was performed using a 16S PCR targeted by universal primers [31]. The quantitative PCRs were run on LightCycler 1.5 or 2.0 (Roche, Almere, the Netherlands). LightCycler software (Version 4.1) resulted in the calculation of the number of S. pneumoniae DNA copies of the positive control.