Fig 8. Detection of B. pseudomallei and B. mallei by qPCR.

(A) Real-time qPCR detection of bucl16-gene target. Genomic DNA of 25 B. pseudomallei (red) and 15 B. mallei strains (blue), and control DNA from 4 B. thailandensis, 4 B. cenocepacia, and 6 B. multivorans strains (gray). (B) qPCR detection of bucl16 target in the presence of human plasma and in spleen extracts from infected mice. 25 ng of gDNA from Bp K96243 was used as a positive control (blue line). Amplification of bucl16 in qPCR reaction spiked with 5% human plasma is shown (green line). Mice were infected with Bp HBPUB10134a and CFU counts used in each qPCR reaction were based on plating spleen extracts on blood agar. Positive amplification is shown for spleen samples with 5x10⁴ CFU (red lines: square, undiluted; triangle, 1:10 dilution; circle, 1:100 dilution; diamond, 1:1000 dilution) and 5x10³ CFU (gray lines: square, undiluted; triangle, 1:10 dilution), while no amplification was obtained for crude spleen samples with original 2x10² CFU and 10 CFU per reaction and no template control, NTC (black lines). Inset; amplification of bucl markers 5, 13, 14, and 16 by standard PCR using crude spleen samples containing 5x10⁴ CFU per reaction. M, 50-bp DNA ladder.