16S rRNA in the hospital environment: Object surface
The first round of PCRs was conducted using the following program: 3 min of denaturation at 95, 25 cycles of 30 s at 95, 30 s for annealing at 55, 30 s for elongation at 72, and a final extension at 72 for 5 min. PCRs were performed in a 25 L mixture containing 12.5 L of 2KAPA HiFi HotStart ReadyMix, 1 L of each primer (1 M), and 12.5 ng of template DNA. AMPure XP beads were used for post-PCR purification. The second round of PCRs were conducted using the following program: 3 min of denaturation at 95, 8 cycles of 30 s at 95, 30 s for annealing at 55, 30 s for elongation at 72, and a final extension at 72 for 5 min. PCRs were performed in 50 L mixture containing 25 L of 2KAPA HiFi HotStart ReadyMix, 5 L of each primer, 10 L water and 5 L of template DNA from products purified in the previous step. AMPure XP beads were used again for purification. Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocol provided by Majorbio Bio-Pharm Technology Co. Ltd.