Modification of the multiplex plasmid PCR assay for Borrelia miyamotoi strain LB-2001 based on the complete genome...

The genome of Borrelia spp. consists of an approximate 1 megabase chromosome and multiple linear and circular plasmids. We previously described a multiplex PCR assay to detect plasmids in the North American Borrelia miyamotoi strains LB-2001 and CT13-2396. The primer pair sets specific for each plasmid were derived from the genome sequence for B. miyamotoi strain CT13-2396, because the LB-2001 complete sequence had not been generated. The recent completion of the LB-2001 genome sequence revealed a distinct number of plasmids (n = 12) that differed from CT13-2396 (n = 14). Notable was a 97-kilobase plasmid in LB-2001, not present in CT13-2396, that appeared to be a rearrangement of the circular plasmids of strain CT13-2396. Strain LB-2001 contained two plasmids, cp30-2 and cp24, that were not annotated for strain CT13-2396. Therefore, we re-evaluated the original CT13-2396-derived multiplex PCR primer pairs and determined their location in the LB-2001 plasmids. We modified the original multiplex plasmid PCR assay for strain LB-2001 to include cp30-2 and cp24. We also determined which LB-2001 plasmids corresponded to the amplicons generated from the original CT13-2396 primer sets. These observations provide a more precise plasmid profile based on the multiplex PCR assay and reflect the complexity of gene rearrangements that occur in B. miyamotoi strains isolated from the same geographic region.