Retroscript kit

Purified RNA (1.0 µg) was subjected to reverse transcription with random decamers using a RETROscript kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The resulting reverse transcriptase (RT) mixtures were used as the template for PCRs with internal primers BLA1-5 and BLA1-6 for bla1, BLA2-5 and BLA2-6 for bla2, and 16S-1 and 16S-2 for the 16S rRNA housekeeping gene, which were previously described by Chen et al. (10 (link)). Semiquantitative PCRs were carried out with a RETROscript kit as described by the manufacturer with 2 U of SuperTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) in 50-μl reaction mixtures containing 100 ng of RT mixture template. PCR amplification conditions were as follows: 1 cycle of denaturation at 94°C for 4 min, followed by 20 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s and template extension at 72°C for 1 min, and 1 cycle of final extension at 72°C for 5 min. Genomic DNA extracted from B. anthracis Sterne was used as a positive control. A no-RT sample and a no-template sample were included as negative controls for each reaction set. Amplification products were detected by gel electrophoresis on a 1% agarose gel, and size was assessed with a DNA ladder (Invitrogen low-DNA mass ladder; Thermo Fisher Scientific, Waltham, MA, USA).