Bacterial 16S rRNA Amplification and Identification

The total genomic DNA of the bacterial isolates was extracted using the Bacterial DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) according to its manufacturer’s instructions. The 16S rRNA gene fragment was amplified using the primers 27F and 1492R [37 ]. The PCR reaction cycling condition was as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, and final elongation at 72 ℃ for 10 min. PCR products were purified and sent to Qinke (Qinke Company, Xi’an, China) for sequencing by the Applied Biosystems 3730XL (ABI 3730XL) sequencer. With the sequencing results, the strains were identified using the EzTaxon-e server [38 (link)]. The neighbor-joining trees were recovered in MEGA 6.0 [39 (link)]. The sequences were aligned with ClustalW [40 (link)]. Kimura’s two-parameters model was utilized to calculate evolutionary distances [41 (link)]. Confidence in the topologies of the resultant tree was calculated by the bootstrap test with 1000 resamplings [42 (link)].