MLST genotyping

It has been performed concerning the previously described method (Zhuang, 2015). The program of nine genomes was designed according to Whatmore et al., including 7 housekeeping genes, 1 outer membrane protein gene, and 1 intergenic fragment (Whatmore et al., 2007). The primer sequences of the MLST nine loci are shown in Table 2. Genomic DNA of vaccine strains, including B. melitensis M5, B. abortus A19, and B. suis S2, were used as positive controls to monitor PCR protocol. The PCR cycling parameters of MLST were as follows: 95°C for 5 min, followed by 30 cycles of 94°C for the 30 s, 63°C for 30 s, 72°C for 60 s, and an elongation at 72°C for 10 min. Then, the products were sent to the Biotechnology company for sequencing. Each gene sequence was entered into the MLST online database (https://pubmlst.org/bigsdb?db=pubmlst_brucella_seqdef&page=batchSequenceQuery) to specify its defined value. Subsequently, the profile of the nine genes was identified as a specific sequence type (ST) based on MLST online database. The ST genotypes of the Brucella were marked on the map of Guizhou Province for analysis of their distribution characteristics. The Web-based MLST database [https://pubmlst.org/bigsdb?db=pubmlst_brucella_seqdef&page=Download alleles (pubmlst.org)] was utilized to collect MLST genotypes of Brucella from different countries. A minimum spanning tree (MST) was constructed using BioNumerics software (version 8.0) based on ST data and an unweighted arithmetic mean algorithm (UPGMA) to show genetic relationships between 83 Brucella isolates and representative strains of Brucella from China and other countries.

The primers sequence of Brucella MLST, MLVA-16, and rpoB genotyping schemes.