Bacterial Identification via 16S rRNA Sequencing

The streak plate technique was employed to obtain the pure culture, and then the microbial classification was carried out according to the colony morphology. 16S rRNA gene sequence was used for the identification of bacterial strains. TIANamp Bacteria DNA Kit (China, TIANGEN) was used to extract the DNA. The extracted DNA of bacteria was used as the template to carry out the polymerase chain reaction (PCR), and P0 and P6 [34 (link)] (Table 1) were used as primers to amplify the 16S rRNA gene of bacteria. The total PCR reaction mixture was 25 μ L, i.e., 10× Taq Buffer 2.5 μ L, dNTP Mixture (2.5 mmol/L) 0.5 μ L, MgCl₂ 1.5 μ L, primer P0 0.5 μ L, primer P6 0.5 μ L, Taq enzyme (5 U / μ L) 0.3 μ L, template (total genomic DNA) 2 μ L, sterile water 17.2 μ L. The PCR amplification conditions were as follows:
$$95{}^{\circ }\mathrm{C}\left(1,,min,,30,,\mathrm{s}\right)-\left[95,,{}^{\circ },\mathrm{C},,\left(1,,min,,30,,\mathrm{s}\right),,-,,56,{}^{\circ },\mathrm{C},,\left(30,,\mathrm{s}\right),,-,72,{}^{\circ },\mathrm{C},,\left(1,,min,,30,,\mathrm{s}\right)\right]\left(25,,\mathrm{cycles}\right)-72{}^{\circ }\mathrm{C}\left(10,,min\right)-4{}^{\circ }\mathrm{C}$$
Table 1

Primers sequence

Primers	Seqence
P0	5′-GAGAGTTTGATCCTGGCTCAG-3’
P6	5′-CTACGGCTACCT TGTTACGA-3’

The full-length sequence of 16S rRNA gene was sequenced by pyrophosphate sequencing and spliced. In the analysis and comparison of the microbial sequence, only those with 100% consistent sequence are considered to be the same microorganism. To identify the microbial species, the full-length base sequences of 16S rRNA gene were compared in the NCBI database. Researchers generally believe that the similarity of bacterial 16S rRNA below 98.7% can be considered a new bacterial species, and less than 94.50% can be considered a new bacterial genus [35 (link)].
The 16S rRNA sequences of all strains obtained were uploaded to NCBI GenBank, and the registration numbers are listed in the Supplementary Information Tables.