DNA Extraction and PCR Amplification

The target DNA of all the isolates were extracted using a QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The sequences of all the serogroup 1-specific and subgroup-specific primers used are listed in Supplementary Table S1. The “lag-1” primers were used for amplification of the internal lag-1 fragment in all the stains. The “lag-1” primer pairs were used as “consensus” primers for all the strains. According to a previous study, lag-1 can be subtyped into “lag-1 Philadelphia,” “lag-1 Knoxville,” and “lag-1 Allentown” (Thurmer et al., 2009). The “intergenic region ORF 6–8,” “intergenic region ORF 7–9,” primers were used for amplification of “subgroup Benidorm/Bellingham” and “subgroup Knoxville” fragments, respectively, in the previous study (Thurmer et al., 2009). And in this study we named “intergenic region ORF 6–8,” “intergenic region ORF 7–9,” primers as “ORF 7,” “ORF 8,” respectively (listed in Supplementary Table S1). And the loci of “ORF 7,” “ORF 8” were shown in a schematic representation (Supplementary Figure S1). All PCRs were performed in thermal cyclers (SensoQuest LabCycler, Senso, Germany or LifeECO Thermal Cycler, BIOER, China) using Quick Taq HS DyeMix (Toyobo, Japan) by the following cycling parameters: denaturation at 94°C for 2 min; followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min; and a final extension at 68°C for 7 min. The PCR products were resolved by 2.0% agarose gel electrophoresis and stained with GoldView or ethidium bromide, visualized under UV light, and analyzed using a Gel Doc system (BioRad, CA, USA).