TABLE 2.
PCR and qRT-PCR primers and probes used in this study
| Primer name | Sequence (5′–3′)a | Purpose or description | Amplicon size(s) (bp) | Source |
|---|---|---|---|---|
| 1924_up_F | GCGGCACCACGTATATCAATAAAA | Amplify upstream region of pilA1 (PD1924) | 981 | This study |
| 1924_up_R | GCAACACCTTCTTCACGAGGCAGACAGAGAATTCATGTGTGGGGGTTTA | |||
| 1924_dn_F | GAGATTTTGAGACACAACGTGGCTTATGAATACACACAGCAACACGATC | Amplify downstream region of pilA1 (PD1924) | 1,190 | This study |
| 1924_dn_R | TTGGAGAAGAGGCGTGTTAAAAAC | |||
| 1924_int_F | ATATACAGGGTGCTTGCTGATTGA | Amplify internal region of pilA1 construct | 2,952 | This study |
| 1924_int_R | GGATGGGTTTAGGGATGCTGATAA | |||
| 1924_out_F | AAACCACCACCGATAACAACAATC | Confirm pilA1 mutants (primers target outside the pilA1 construct) | 3,509, 2,884b | This study |
| 1924_out_R | AATAGCGTTGGTAAGAAATCCAGC | |||
| 1926_up_F | CATTTCACTTTGACTTCACCCGAA | Amplify upstream region of pilA2 (PD1926) | 1,032 | This study |
| 1926_up_R | TGCCCCGTATTCAGTGTCGCTGATTAATAGCGTTGGTAAGAAATCCAGC | |||
| 1926_dn_F | GCCTGGTGCTACGCCTGAATAAGTGAAACACGATTCATGGGTAAATGCTC | Amplify downstream region of pilA2 (PD1926) | 700 | This study |
| 1926_dn_R | TGAGCGTCAATTTTAGAGGATGGA | |||
| 1926_int_F | TCAGCAATACTCATACTGGCACTT | Amplify internal region of pilA2 construct | 2,773 | This study |
| 1926_int_R | AACGTGTGCTTGAATCTTCGAATT | |||
| 1926_out_F | ACAAGAGTGAGCCGTTACAACTAT | Confirm pilA2 mutants (primers target outside the pilA2 construct) | 3,004, 2,352b | This study |
| 1926_out_R | CTTTTCCAATGAGCAGTTATCGGG | |||
| Kan_F | GTCTGCCTCGTGAAG | Amplify kanamycin cassette | 1,204 | This study |
| Kan_R | AAGCCACGTTGTGT | |||
| Cm_F | AATCAGCGACACTGAATACGG | Amplify chloramphenicol cassette | 1,119 | This study |
| Cm_R | TCACTTATTCAGGCGTAGCAC | |||
| pilA2C_up_F | CGCGCCCGTTATTAATCGAA | Amplify upstream fragment of pilA2 complementation construct (contains upstream of NS1 region and kanamycin cassette) | 1,957 | This study |
| pilA2C_up_R2 | ATATTGAAGGGTGCAATACAAAGCATCTAGTCTCAACCATCATCGATGAA | |||
| pilA2C_dn_F2 | GTTTCAAGAGAGAGAGCGTTCAACACGATGCTGTTAACCATTGTCATC | Amplify downstream fragment of pilA2 complementation construct (this is same as NS1 downstream fragment) | 799 | This study |
| pilA2C_dn_R | TAACCTTGTCAGCGTAGATG | |||
| pilA2_F | ACAATTCATCGATGATGGTTGAGACTAGATGCTTTGTATTGCACCCTTCAAT | Amplify pilA2 coding region, 237 bp upstream, and 216 bp downstream to include promoter and terminator regions | 950 | This study |
| pilA2_R | GCCATTGATGACAATGGTTAACAGCATCGTTTCAAGAGAGAGAGCGTTCAAC | |||
| pilA_F | AAACACCGGACTTGCCAACATCAC | Primer and probe to amplify fragment of pilA1 | 140 | 30 |
| pilA_R | TGTTGCATGTCCACTGACCTCCAT | |||
| pilA_Pc | AAACCATCGCTTGGAATCGTAGCGTCGA | |||
| PD1926_F | CACTCCTAACGCTATTGGACTAC | Primer and probe to amplify fragment of pilA2 | 117 | 32 |
| PD1926_R | TTGACCTGACCATTACCAATCA | |||
| PD1926_Pc | TGGTGGACATCACAACTACTGGCG | |||
| nuoA_F | AGACGCACGGATGAAGTTCGATGT | Reference gene for qRT-PCR | 30 | |
| nuoA_R | ATTCCAGCGCTCCCTTCTTCCATA | |||
| nuoA_Pc | TTCATCGTGCCTTGGACTCAGGTGTT | |||
| 1691_up F | AGGCAACCTGACAGCGATAC | Amplify upstream region of 1691 construct | 541 | This study |
| 1691_up_R2 | GCAACACCTTCTTCACGAGGCAGACCGTTGATGTTCGAGAAGTGCG | |||
| 1691_Dn_F2 | GAGATTTTGAGACACAACGTGGCTTTGCATCAACCCCAAAGCTGA | Amplify downstream region of 1691 construct | 504 | This study |
| 1691_Dn R | AAGCGATCCAATGAAGGGCT | |||
| 1691_int_F | ACGGCCCTTCTCTAAGATTGC | Amplify internal region of 1691 construct | 2,152, 987b | This study |
| 1691_int_R | TGTGTGGTGCTTGCATATTCTG | |||
| 1695_up_F | CGATGGCCTGTTGATAGCGATA | Amplify upstream region of 1695 construct | 1,027 | This study |
| 1695_up_R | CCGTATTCAGTGTCGCTGATTGTCACCACGTTTGAGGAGTTTG | |||
| 1695_Dn_F | GTGCTACGCCTGAATAAGTGAAACGGGGTGAATGGACATTAG | Amplify downstream region of 1695 construct | 1,018 | This study |
| 1695_Dn_R | CAGATGGGGAGTGCTGCTTTA | |||
| 1695_int_F | AAAGACGAAATCCTGGAGCTGTAT | Amplify internal region of 1695 construct | 2,640, 1,546b | This study |
| 1695_int_R | TTGATACCAATTGGAAGACAACGC |
a
Underlining indicates the 5′ extended region of the primer that is homologous to one of the antibiotic resistance cassettes to facilitate fusion of chromosomal sequence to a selectable marker by overlap extension PCR.
b
Amplicon size in the wild-type strain without the insertion of antibiotic resistance gene.
c
These probes were used for reverse transcription-quantitative PCR (qRT-PCR) and were labeled with 6-carboxyfluorescein (FAM) at the 5′ end and Black Hole 588 Quencher-1 (BHQ1) at the 3′ end.