TABLE 2.
Oligonucleotide primers used in this work
| Primer | Sequencea | Position | Aplication |
|---|---|---|---|
| PM0298intup | 5′-TTACCTGACGAGTTTGTTCG-3′ | +45b | Upper primer to detect the present of the single transcrit in RT-PCR experiment; also used as upper primer to obtain the 169-bp internal fragment of P. multocida ORF PM0298 |
| PhgbArp | 5′-TCTAGAACTATCCGCCAAAATGGCC-3′ | +1079b | Lower primer to detect the present of the single transcrit in RT-PCR experiment; also used as lower primer to obtain the 360-bp P. multocida hgbA 5′ end |
| PPM0298up | 5′-GAGCATCAAATTAGGTCTG-3′ | −337b | Upper primer to obtain a fragment containing the P. multocida PM0298-PM0299-hgbA operon |
| HgbAextrp | 5′-CGCTAGCCGATCTCTAATCC-3′ | +4026b | Lower primer to obtain a fragment containing the P. multocida PM0298-PM0299-hgbA operon |
| NdehgbA | 5′-CATATGCGTACACAACAACAATAAAAATTTCTGC-3′ | +1001b | Upper primer to obtain the 3-kb fragment used to overexpress the P. multocida hgbA gene |
| NothgbA | 5′-GCGGCCGCCGCTAGCCGATCTCTAATCC-3′ | +4026b | Lower primer to obtain the 3-kb fragment used to overexpress the P. multocida hgbA gene |
| HgbAintup | 5′-CTGAACTTGATACGATTACC-3′ | +1094b | Upper primer to obtain the 832-bp internal fragment of the P. multocida hgbA gene |
| HgbAintrp | 5′-CCAAAATGGCGTAACAG-3′ | +1926b | Lower primer to obtain the 832-bp internal fragment of the P. multocida hgbA gene |
| HgbArp | 5′-CGGTTTAATATAACCAACC-3′ | +3728b | Primer to confirm disruption of the P. multocida hgbA gene by insertion of the pUA967 plasmid |
| PM0298intrp | 5′-GTTCTGGAAATAGTTGACGC-3′ | +214b | Lower primer to obtain the 169-bp internal fragment of P. multocida ORF PM0298 |
| PM0299intup | 5′-TCGGTGAAGATGGTAATCCC-3′ | +538b | Upper primer to obtain the 258-bp internal fragment of P. multocida ORF PM0299 |
| PM0299intrp | 5′-TTGCTTTGAGTGCCTCAACG-3′ | +796b | Lower primer to obtain the 258-bp internal fragment of P. multocida ORF PM0299 |
| PhgbAup | 5′-TCTAGAATCTTTTGATGCGGTTGCGCG-3′ | +719b | Upper primer to obtain the 360-bp containing the P. multocida hgbA 5′ end |
| Aad | 5′-CGGCGATCACCGCTTCCC-3′ | +2c | Plasmid primer to confirm disruption of P. multocida ORFs PM0298 and hgbA by insertion of the pUA965 and pUA967 plasmids, respectively |
| RTHgbAup | 5′-TATTGTGGGGTCATTTTGGCG-3′ | +1052b | Upper primer to detect hgbA transcript |
| RTHgbArp | 5′-TGAATATCTTCTTTCACTGGG-3′ | +2343b | Lower primer to detect hgbA transcript |
| RecAup | 5′-GCTCTATTATGAAATTGGGCG-3′ | +100d | Upper primer to test PM1078 RNA integrity |
| RecAdw | 5′-CTAACCATTTCATCGCG-3′ | +957d | Lower primer to test PM1078 RNA integrity |
a
When present, added restriction sites are underlined.
b
Position of the 5′ end of the oligonucleotide with respect the translational start point of P. multocida ORF PM0298.
c
Position of the 5′ end of the oligonucleotide with respect the translational start point of the aad gene of the pUA826 plasmid.
d
Position of the 5′ end of the oligonucleotide with respect the translational start point of the P. multocida recA gene.