Fig. 2
Optimisation of the method for isolation of DNA and RNA from throat swabs in eNat™. a Pre-lysis versus No pre-lysis extractions using the combined RNA + DNA approach. eNats™ were spiked with S. pyogenes M75 at three concentrations and 200 μl aliquots (1.8 × 102, 1.8 × 104, 1.8 × 106 total GE) underwent DNA extraction using a chemical Pre-lysis step (●) or No pre-lysis (■). A larger 600 μl aliquot (3x vol) of eNat™ medium spiked with M75 (5.4 × 104 total GE) was processed without pre-lysis (◆). Data shown in total GE to account for differences in input bacteria. Each point represents the mean and SD of two eNats™ tested in duplicate qPCRs. b eNats™ containing a range of M75 were used to test separate column extractions (RNA-only ■ or DNA-only ●) versus combined RNA + DNA isolation (RNA⃞ and DNA❍). The emm75 qPCR was used to assess DNA whilst RNA was converted to cDNA and tested in a SYBR Green qPCR for the housekeeping gene, gyrA. Each point represents the mean and SD of three eNats™ tested in duplicate qPCRs targeting the emm75 gene