Table 3. Criteria for detection of Mycoplasma spp., M. bovis, and A. laidlawii in milk with the multiplex qPCR assay.
| Organismb | Gene target (Cut-off Ct value)a | ||
|---|---|---|---|
| 16S rRNA (Ct ≤ 32) |
rpoB (Ct ≤ 33) |
ITS (Ct ≤ 32) |
|
| Mycoplasma spp. | + | – | – |
| M. bovis c | + | + | – |
| A. laidlawii | – | – | + |
Notes:
a
Ct, Threshold Cycle value; positive (+) and negative (−) symbols indicate a positive and negative result for that group of bacteria by the multiplex qPCR assay.
b
M. californicum ATCC33461 M. bovis ATCC25523, and A. laidlawii ATCC23206 were used for determining LOD levels of Mycoplasma, M. bovis, and A. laidlawii, respectively.
c
M. bovis gDNA was expected to be detected by both the 16S rRNA and rpoB assays, therefore a positive M. bovis sample is expected to have <3 Ct value difference for those targets.