One lozenge from each of groups A and B was dissolved separately in 1 mL of sterile, PCR-grade water. Aliquots of the clinical samples and the dissolved lozenges were mechanically lysed using Lysing Matrix E bead tubes and RLT Plus lysis buffer for 30 s × 2 using the Omni Bead Ruptor 24. Purification of genomic DNA was achieved with the AllPrep DNA/RNA Isolation Kit (Qiagen, NRW, Germany), using a spin column for DNA isolation. PCR-grade water was used as a negative extraction control.
The V3-V4 region of the bacterial 16S rRNA gene was amplified using Illumina-compatible primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-2135 (link), as previously described32 (link). Duplicate PCR reactions were pooled and purified for sequencing, as previously described29 (link). Normalised samples were submitted to Auckland Genomics Ltd for sequencing on the Illumina MiSeq platform.
The V3-V4 region of the bacterial 16S rRNA gene was amplified using Illumina-compatible primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-2135 (link), as previously described32 (link). Duplicate PCR reactions were pooled and purified for sequencing, as previously described29 (link). Normalised samples were submitted to Auckland Genomics Ltd for sequencing on the Illumina MiSeq platform.
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