Figure 2. Sensitive and specific detection of individual BbITS with PCR primers for each unique sequence tag.

The specificity of each PCR primer pair for detection of a single BbITS clone when amplified from a mixed DNA sample was demonstrated as shown in (A). Seven mixed templates were generated in which the genomic DNAs of 6 BbITS clones were combined in equal proportions, with a single clone omitted in succession, as indicated at the top of panel A. The amount of DNA used per PCR reaction was the equivalence of 2.4×10⁷ spirochetes total, or 4×10⁶ per clone. All 7 BbITS primer pairs, as identified on the left side of panel A, were tested with all 7 mixed samples. The sensitivity of each PCR primer pair for an individual BbITS clone when amplified from a mixed DNA sample was determined as shown in (B). 10-fold serial dilutions were prepared with genomic DNA for each individual BbITS clone, as indicated at the top of panel B. Seven mixed DNA templates were prepared from these dilution series with equal volumes of undiluted DNA from the remaining 6 clones. The dilution series spanned the range from all 7 BbITS clones present at the same amount (2.4×10⁷ total spirochetes), to an extreme of 1∶10⁻⁶ for the diluted clone relative to the other 6 BbITS. Each BbITS primer pair, as identified on the left side of panel B, was tested with the dilution series of its specific target DNA in the presence of undiluted DNA from the other 6 BbITS.