DNA from microscopy-quantified blood-stage parasites [33 ] was extracted from 5 µl of blood using a semi-automatic Kingfisher Flex Magnetic Particle Processor and MagMAX™-96 DNA Multi-Sample Kit (Thermo Fisher Scientific, Waltham, MA, USA) as per [34 (link)], and was frozen at − 20 °C until use. These blood-stage DNA samples were used to determine qPCR efficiency and the limit of detection (LOD).
DNA was extracted from head/thorax mosquito samples and feeding substrates following the CTAB-based phenol–chloroform extraction method of Chen et al. [35 (link)] with minor modifications (as described in Schneider et al. [33 ]). Extracted DNA was eluted in 30 µl (mosquitoes, supplemented feeding substrates) or 16 µl (mosquito expectorate substrates) of water and frozen at − 20 °C until use. DNA extracts from mosquitoes, but not from feeding substrates, were diluted fourfold to reduce the effect of inhibitors originating from mosquito material on the performance of the PCR. All PCR reactions were run using 7 µl of (diluted) DNA extracts, and data are presented as genomes/PCR, unless stated otherwise, to account for differences in sample processing.
DNA was extracted from head/thorax mosquito samples and feeding substrates following the CTAB-based phenol–chloroform extraction method of Chen et al. [35 (link)] with minor modifications (as described in Schneider et al. [33 ]). Extracted DNA was eluted in 30 µl (mosquitoes, supplemented feeding substrates) or 16 µl (mosquito expectorate substrates) of water and frozen at − 20 °C until use. DNA extracts from mosquitoes, but not from feeding substrates, were diluted fourfold to reduce the effect of inhibitors originating from mosquito material on the performance of the PCR. All PCR reactions were run using 7 µl of (diluted) DNA extracts, and data are presented as genomes/PCR, unless stated otherwise, to account for differences in sample processing.
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