The qPCR reactions were performed in 30 μl volume containing 2.3 μl of 20 mg/ml bovine serum albumin (BSA; Sigma A2153), 15.05 μl SensiFAST Probe Lo-ROX Mix (Bioline BIO84005), 3.05 μl forward primer (5 pmol/μl), 3.05 μl reverse primer (5 pmol/μl) and 1.55 μl TaqMan probe (5 pmol/μl), and 5 μl of DNA extract. The primers and probes used were as follows:
For B. anthracis, the chromosomal marker targeting prophage lambdaBa03 (PL3; Wielinga et al., 2011 (link)),
PL3_F: AAAGCTACAAACTCTGAAATTTGTAAATTG.
PL3_R: CAACGATGATTGGAGATAGAGTATTCTTT.
Tqpro_PL3: FAM-AACAGTACGTTTCACTGGAGCAAAATCAA-BHQ-1.
For Y. pestis, the gene capR encoding the Lon ATP-dependent serine protease (Steinberger-Levy et al., 2016 (link)),
capF: GGATTACGATCTCTCGGATGTGA.
capR: AGCCGGACAGACGAATAACTTC.
Taq-CapR: FAM-TTGTGGCGACCTCTAACTCCATGAATATTCC-BHQ-1.
For F. tularensis, the gene fopA encoding for an outer membrane protein (Versage et al., 2003 (link)),
fopAF: ATCTAGCAGGTCAAGCAACAGGT.
fopAR: GTCAACACTTGCTTGAACATTTCTAGATA.
fopAP: FAM-CAAACTTAAGACCACCACCCACATCCCAA-BHQ-1.
The PCR thermal conditions were as follows: 3 min at 60°C followed by 40 cycles of 15 s at 95°C and 35 s at 60°C.
For B. anthracis, the chromosomal marker targeting prophage lambdaBa03 (PL3; Wielinga et al., 2011 (link)),
PL3_F: AAAGCTACAAACTCTGAAATTTGTAAATTG.
PL3_R: CAACGATGATTGGAGATAGAGTATTCTTT.
Tqpro_PL3: FAM-AACAGTACGTTTCACTGGAGCAAAATCAA-BHQ-1.
For Y. pestis, the gene capR encoding the Lon ATP-dependent serine protease (Steinberger-Levy et al., 2016 (link)),
capF: GGATTACGATCTCTCGGATGTGA.
capR: AGCCGGACAGACGAATAACTTC.
Taq-CapR: FAM-TTGTGGCGACCTCTAACTCCATGAATATTCC-BHQ-1.
For F. tularensis, the gene fopA encoding for an outer membrane protein (Versage et al., 2003 (link)),
fopAF: ATCTAGCAGGTCAAGCAACAGGT.
fopAR: GTCAACACTTGCTTGAACATTTCTAGATA.
fopAP: FAM-CAAACTTAAGACCACCACCCACATCCCAA-BHQ-1.
The PCR thermal conditions were as follows: 3 min at 60°C followed by 40 cycles of 15 s at 95°C and 35 s at 60°C.
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