Table 2

List of validation results from real-time PCR assays

ParameterResult by assay
TTS1-orf2TTS1-orf11lpxO8653mprAYLF/BTFC266152
Analytical specificity, % (no. positive/total)
    B. pseudomallei strains100 (365/365)100 (365/365)a100 (365/365)100 (365/365)100 (365/365)99.7 (364/365)100 (365/365)
    Nontarget strains100 (0/115)99 (1/115)b87.8 (14/115)c100 (0/115)100 (0/115)100 (0/115)100 (0/115)
LoD (GE/reaction)d5101010505e10
Diagnostic sensitivity, % (no. positive/total)80 (40/50)70 (35/50)76 (38/50)68 (34/50)54 (27/50)80 (40/50)68 (34/50)
Diagnostic specificity, % (no. positive/total)100 (0/22)100 (0/22)100 (0/22)100 (0/22)100 (0/22)95.5 (1/22)f100 (0/22)
  • a The TTS1-orf11 assay detected one of the B. pseudomallei strains only at a high CT value of 36.9.

  • b The TTS1-orf11 assay provided a weak false-positive result for 1/23 B. mallei strains at a CT value of 37.4.

  • c The lpxO assay provided false positive results for 14/23 B. mallei strains.

  • d Shown is the number of B. pseudomallei genome equivalents (GE) per PCR. One B. pseudomallei genome equivalent equals approximately 7.8 fg based on a genome size of 7.2 Mb.

  • e This LoD refers to a B. pseudomallei genome containing the YLF locus.

  • f This likely reflects a contamination event as the weak false-positive result (CT value of 37.7) was from a buffy coat sample from a healthy volunteer, and weak positive results above the cutoff of 40 CT values were also evident for this sample with other assays.