16S- dental spitoon biofilm
The V4 hyper-variable region of the 16S-rRNA gene was amplified in duplicates, using the forward primer 16s-515F (GTGCCAGCMGCCGCGGTAA) and the reverse primer 16s-806R (GGACTACVSGGGTATCTAAT) to cover both bacteria and archaea.PCR was conducted using the HotStarTaq Plus Master Mix Kit (QIAGEN) and carried out as follows: initial denaturation at 95C for 5 minutes, denaturation at 95C for 30 sec, annealing at 53C for 40 sec, and extension at 72C for 1 min for 30-35 cycles, followed by final elongation at 72C for 10 min. Paired-end sequencing (bTEFAP) was performed by Molecular research DNA (Shallowater, Texas, USA; http://www.mrdnalab.com) on an Illumina MiSeq sequencing platform (Illumina, San Diego, California, USA) following the manufacturer's instructions.