Bacterial Colonization of Textile Microplastics in River Water and the Distribution of Antibiotic Resistance Genes
Amplification of the V4 region of the 16S rRNA gene was performed using primers 515F (5GTGYCAGCMGCCGCGGTAA3) and 806R (5GGACTACNVGRGTWTCTAAT3), following the method of Caporaso et al., with slight modification. In brief, the temperature sequence for the PCR cycle conditions for the first PCR was as follow: 95 C for 3min; 10 cycles at 98 C for 20s, 50 C for 15s, and 72 C for 45s; with a final extension at 72 C for 1min. For the second PCR, using fusion primers and the first PCR product as a template: 95 C for 3min; 35 cycles at 98 C for 20s, 50 C for 15s, and 72 C for 45s; with a final extension at 72 C for 1min. Finally, the concentration of purified PCR product was measured using a Qubit 2.0 fluorometer. Barcode-tagged amplicons for the different samples were then mixed in equimolar concentrations. Sequencing of bacterial amplicons was performed on the Genexus IonTorrent platform.