Table 1.
Oligonucleotide primers, PCR protocols, and amplicon sizes for each PCR assay used in this study.
| Bacterial species and targeted gene | Primer sequences (5'−3') and PCR cycling conditions | Amplicon size (bp) | References | |
|---|---|---|---|---|
| Mh, lktC- artJ intergenic region | F – GTCCCTGTGTTTTCATTATAAG R – ACTCGATAATTATTCTAAATTAG 95°C, 5 min; (94°C, 30 s; 58°C, 45 s; 72°C, 60 s) × 35 cycles; 72°C, 10 min | 385 | (24) | |
| Pm, 23S rRNA | F – GGCTGGGAAGCCAAATCAAAG R – CGAGGGACTACAATTACTGTAA 95°C, 5 min; (94°C, 30 s; 58°C, 45 s; 72°C, 60 s) × 35 cycles; 72°C, 10 min | 1,432 | (25) | |
| Hs, 16S rRNA | F – GAAGGCGATTAGTTTAAGAG R – TTCGGGCACCAAGTRTTCA 95°C, 5 min; (94°C, 30 s; 55°C, 45 s; 72°C, 60 s) × 35 cycles; 72°C, 10 min | 400 | (26) | |
| Mb, uvrC | F – CCTGTCGGAGTTGCAATTGT R – GCACTGCGCTCATTTAAAGC 95°C, 5 mina; (94°C, 30 s; 61.5°C, 30 s; 72°C, 30 s) × 35 cycles, 72°C, 10 min | 92 | (23) | |
| Mh serotype A1, hypothetical protein | F – CATTTCCTTAGGTTCAGC R – CAAGTCATCGTAATGCCT | 95°C, 15 min; (94°C, 30 s; 55°C, 45 s; 72°C, 1 min) × 35 cycles; 72°C, 10 min | 306 | (27) |
| Mh serotype A2, core-2/I-branching enzyme | F – GGCATATCCTAAAGCCGT R – AGAATCCACTATTGGGCACC | 160 | ||
| Mh serotype A6, TupA | F – TGAGAATTTCGACAGCACT R – ACCTTGGCATATCGTACC | 78 | ||
a
Denaturation time increased from 5 min to 10 min if cell lysis was required during the PCR cycle.
Mh, Mannheimia haemolytica; Pm, Pasteurella multocida; Hs, Histophilus somni; Mb, Mycoplasma bovis; F, forward primer; R, reverse primer.