TABLE 1.
Oligonucleotide primers comprising the multilocus identification scheme
| Gene target | Phage namea | Primer name | Sequence (5′ → 3′) | Gene type | Tmb | Annealing temp | Amplicon size (bp) | Reference or source |
|---|---|---|---|---|---|---|---|---|
| int | Φ24B | Φ24Bint 5′ | GCCAGGCTTTCTGAGCTACG | 24B int | 55.0 | 16 | ||
| Φ24Bint 3′ | GCCTAAAATCATGCGTTCTCC | 55.0 | 16 | |||||
| lambda, H19J, | 1F | GTTACMGGGCARMGAGTHGG | 1 int | 50.0 | 5 | |||
| HKO22, P434 (group 1) | 1R | ATGCCCGAGAAGAYGTTGAGC | 5 | |||||
| P21, e14 (group 2) | 2F | GTTACTGGWCARCGKTTAGG | 2 int | 50.0 | 5 | |||
| 2R | GATCATCATKRTAWCGRTCGGT | 5 | ||||||
| ST64B (group 3) | 3F | AATGGARATWKCYTATYTVTGTGC | 3 int | 50.0 | 5 | |||
| 3R | TCRTARTCTGARATYCCYTTBGC | 5 | ||||||
| P27, 933W, Fels1, BP4795, Gifsy2, EH297 (group 4) | 4F | CTBGCMTGGGARGATATHGA | 50.0 | 5 | ||||
| 4R | GMCCAGCABGCATARGTRTG | 5 | ||||||
| P4, HK620, Sf6 (group 5a) | 5AF | TGGRAKRAMKTCGAYTTYGA | 5A int | 50.0 | 5 | |||
| 5AR | CAGTTGCMYYTCWATMGCGTCA | 5 | ||||||
| P4, epsilon 15 | 5BF | TWGTKCGTWMMAGTGAATT | 5B int | 50.0 | 5 | |||
| (group 5b) | 5BR | TKGWTRTATRCCGCWCGYAC | 5 | |||||
| Phi80, Gifsy1 | 5CF | GGRMARTYATAAAACKSG | 5C int | 50.0 | 5 | |||
| (group 5c) | 5CR | TGCCCGAGCAKCWTYTCA | 5 | |||||
| P2, L413C, Wphi | 6AF | CTGAGYACWGGAGSAMGWTGG | 6A int | 50.0 | 5 | |||
| (Group 6a) | 6AR | CCBCCRTTMATCATRAARTG | 5 | |||||
| Fels2, PSP3, P186 | 6BF | TVGCWACYGGCGCMMGRTGG | 6B int | 50.0 | 5 | |||
| (Group 6b) | 6BR | CCBCCRTTMATCATRAARTG | 5 | |||||
| P22, SfV, SfII, | 7F | AACATYATMAAYCTKGARTGGCA | 7 int | 50.0 | 5 | |||
| ST64T | 7R | CGAACCATTTCKATRGACTCCCA | 5 | |||||
| P1 | 8F | TGCTTATAACACCCTGTTACGTAT | 8 int | 50.0 | 5 | |||
| 8R | CAGCCACCAGCTTGCATGATC | 5 | ||||||
| cIII | All listed | cIII 5′ | ATGCAATATGCCATTGCA | cIII | 43.0 | 53.0 | 168 | This study |
| cIII 3′ | TTAGTCTGGATAGCCATA | 43.0 | This study | |||||
| N | N (1) 5′ | ATGACACGCAGAACTCAG | N1 | 51.4 | 50.0 | 384 | This study | |
| N (2) 5′ | ATGCAATGCCGAAGCAAC | N2 | 56.5 | 57.5 | 295 | This study | ||
| N 3′ | TYACCTYGCYGTCAGTTG | 54.4 | This study | |||||
| cI | BP4795, H19B | cI (1a) 5′ | ATGGAAAACAAAGATATTCGC | cI 1a | 59.4 | 54.0 | 705 | This study |
| 933W, Stx2(I) | cI (1b) 5′ | ATGGTTCAGAATGAAAAAGTG | cI 1b | 58.1 | 61.0 | 708 | This study | |
| BP4795, H19B, 933W, Stx2(I) | cI (1) 3′ | TCACGAACTTTTCAGCCACTC | 64.2 | This study | ||||
| HK97, Lambda | cI (2a) 5′ | ATGAGCRCAAAAAAGAAACCA | cI 2a | 60.2 | 60.0 | 714 | This study | |
| Nil 2 | cI (2b) 5′ | ATGAAATGGTATGAACTGGCT | cI 2b | 59.9 | 60.0 | 654 | This study | |
| Stx2(II) | cI (2c) 5′ | ATGGATGGTTCCAGTACAGAG | cI 2c | 60.4 | 59.0 | 598 | This study | |
| VT2-Sa | cI (2d) 5′ | GTGGTGTTTAAATACCTTGGT | cI 2d | 57.0 | 59.0 | 510 | This study | |
| HK97, Lambda, Nil 2, Stx2(II), VT2-Sa | cI (2) 3′ | TYAACCAAACGTCTCTTCAGG | cI 2d | 59.3 | 510 | This study | ||
| HK620 | cI (3) 5′ | ATGGAAAATAAAAAATCACTG | cI 3 | 54.9 | 53.0 | 714 | This study | |
| cI (3) 3′ | TCAAACCAGCCTTAGTTTTGT | 61.3 | This study | |||||
| D3112 | cI (4) 5′ | GTGAAATCAGACACTTACGGA | cI 4 | 59.2 | 62.2 | 665 | This study | |
| cI (4) 3′ | CTAAACCATCCAGCGGCTAGC | 66.7 | This study | |||||
| P27 | cI (5) 5′ | ATGAAATCTTTAGGTGAACGC | cI 5 | 59.6 | 62.6 | 723 | This study | |
| cI (5) 3′ | TCAGAAAATATCCCACCTGGC | 64.3 | This study | |||||
| phi 105 | cI (6) 5′ | ATGACTGTAGGGCAAAGAATC | cI 6 | 59.6 | 53.0 | 437 | This study | |
| cI (6) 3′ | GTATTCTTGATCGTCATTTCT | 54.8 | This study | |||||
| cro | HK97, Sakai | Cro (1) 5′ | ATGGAACAACGCATAACCCTG | cro 1 | 65.6 | 59.4 | 201 | This study |
| Cro (1) 3′ | TTATGCAGTTGTTTTTTTGTT | 56.4 | This study | |||||
| HK620 | Cro (2) 5′ | ATGATTCGAATGACACTTGCC | cro 2 | 63.7 | 57.0 | 195 | This study | |
| Cro (2) 3′ | CTATTTGTTTTTCTTGTTGCT | 55.3 | This study | |||||
| 933W, StxII | Cro (3) 5′ | ATGCAAAATCTTGATGAGCCG | cro 3 | 66.0 | 65.0 | 228 | This study | |
| Cro (3) 3′ | TTATGCAGCCAGAAGGTTCTT | 62.6 | This study | |||||
| Sakai, ST64T, VT2Sa | Cro (4) 5′ | ATGAGCAAYCTWCGRAAAWWY | cro 4 | 56.9 | 59.9 | 216 | This study | |
| Cro (4) 3′ | TTARGCRGCWTTRWGYTCMGG | 61.7 | This study | |||||
| P22 | Cro (5) 5′ | ATGTACAAGAAAGATGTTATCGAC | cro 5 | 57.7 | 55.0 | 186 | This study | |
| Cro (5) 3′ | CTTCATGGTTCTTTTGCG | 59.4 | This study | |||||
| ST104 | Cro (6) 5′ | ATGACTAACAAAGCAATACAA | cro 6 | 54.0 | 54.0 | 216 | This study | |
| Cro (6) 3′ | TTAACTTGCTGCCAGTAAGTC | 58.6 | This study | |||||
| SfV | Cro (7) 5′ | ATGAAAGCGTATTGGGACTCT | cro 7 | 61.4 | 54.8 | 201 | This study | |
| Cro (7) 3′ | TTAATCTTTCGGATAGATATC | 55.0 | This study | |||||
| N15 | Cro (8) 5′ | ATGAAACCCGAAGAACTTGTG | cro 8 | 62.8 | 56.0 | 216 | This study | |
| Cro (8) 3′ | CTATTTAGTTCCACTGTTATG CCC | 61.4 | This study | |||||
| BP4795 | Cro (9) 5′ | ATGAGTAATGAACTACTACGCTGG | cro 9 | 60.1 | 56.0 | 234 | This study | |
| Cro (9) 3′ | TTATGCAGCCGATGCTCT | 62.1 | This study | |||||
| cII | All listed | CII - 5′ | ATGRMACRARCAAGYTACAGC | cII | 56.0 | 53.0 | 297 | This study |
| CII - 3′ | TCAGAATTGCATATCAAT | 50.7 | This study | |||||
| Q | All listed | Q ATG 5′ | ATGTTCTTATGGTTCACCG | Q | 53.0 | 55.5 | 435 | This study |
| Q 3′ | TTACGATCGTAAACTATTTTT | 50.0 | This study | |||||
| stx | All sequenced stx-genes | Degen StxA (Type I&II) | TTTGTYACYGTSAYAGCWGAAG | stx | 47.2 | 47.5 | 675 | This study |
| Degen StxB (Type I&II) | TYMTCATTATAYTTDGWRWACT | 49.0 | This study | |||||
| Capsid | CP933X Capsid | CP933X Capsid 5′ | TGGGRCCGGSAWRACATSCTG | capX | 63.3 | 63.3 | 1,572 | This study |
| CP933X Capsid 3′ | TTACGCAGCTCTGCTGTC | 60.48 | This study | |||||
| CP933I Capsid | CP933I Capsid 5′ | ATGACAATCCCAGAACAG | capI | 55.6 | 54.9 | 753 | This study | |
| CP933I Capsid 3′ | TCATACTGCTTTCTCCTT | 51.9 | This study | |||||
| CP933X (3) Capsid | CP933X (3) Capsid 5′ | ATGTCSRTKTACACMACYGCC | capX3 | 55.0 | 58.0 | 1,026 | This study | |
| CP933X (3) Capsid 3′ | TTAYGCCAGYTKKACGGASAC | 58.1 | This study | |||||
| CP933X (2) Capsid | CP933X (2) Capsid 5′ | RTGRCAGCAGAGCTGCGT | capX2 | 63.8 | 55.0 | 1,204 | This study | |
| CP933X (2) Capsid 3′ | TTAMACTGGKGTGTTYARCAA | 52.1 | This study | |||||
| CP933C Capsid | CP933C Capsid 5′ | ATGCMGAGAATAATCGAATTAC | capC | 56.5 | 56.0 | 1,158 | This study | |
| CP933C Capsid 3′ | TTAATCGTCGTCYTSYGGCAG | 63.8 | This study | |||||
| CP933R (3) Capsid | CP933R (3) Capsid 5′ | ATGAAACGAACGCCTGTC | capR3 | 61.3 | 53.0 | 1,590 | This study | |
| CP933R (3) Capsid 3′ | TTACGTCTCACGKGRTGT | 54.6 | This study | |||||
| CP933R Capsid | CP933R Capsid 5′ | ATGGGATTGTTTACGACC | capR | 57.1 | 53.0 | 1,029 | This study | |
| CP933R Capsid 3′ | TTATTTCACCTGTACCAC | 50.0 | This study | |||||
| CP933O/R Capsid 5′ | CP933O/R Capsid 5′ | ATGGTRACGAAAAMATCACTGA | capO/R | 57.5 | 60.6 | 348 | This study | |
| CP933O/R Capsid 3′ | TTACGGCAGCGCYGC | 63.8 | This study | |||||
| P27 Capsid | P27 Capsid 5′ | RYGGYTGATRTTAAAGATGTG | capP27 | 60.0 | 56.9 | 1,224 | This study | |
| P27 Capsid 3′ | AATTTTCAKCAGCTTRATMGC | 57.0 | This study | |||||
| HK620 Major Capsid | HK620 Major Capsid 5′ | ATGGCCTAACAATCTCGA | cap620 | 51.0 | 51.0 | 1,272 | This study | |
| HK620 Major Capsid 3′ | TTACGGATTACCGAAGAA | 49.0 | This study | |||||
| Host recognition proteins | This study | |||||||
| Short-tailed | 933W, VT2-Sa, | VTTF1 5′ | GTTGTTGTTTCGGGGACG | VTTF1 | 64.0 | 55.0 | 1,900 | This study |
| Stx phages | Φ24B | VTUTF 3′ | TCATTCTCCTGTTCTGCC | VTTF2 | 58.8 | This study | ||
| VTTF3 5′ | TGCAGAGGAAAGCTCGAC | VTTF3F | 54.9 | 55.0 | 800 | This study | ||
| VTTF3 3′ | GCAGCCTCTTCTGCCTTT | VTTF3R | 62.3 | This study | ||||
| Long-tailed | P27 TF | P27 (p56) (TF) 5′ | ATGTCTGTAGTGATATCAGGT | TFP27 | 52.4 | 55.3 | 400 | This study |
| Stx phage | P27 (p56) (TF) 3′ | TCATGCCAATCCTCACAA | 61.0 | This study | ||||
| N15 HRP | N15 (TF) 5′ | ATGGCTACATCTACTCCG | TFN15 | 48.0 | 47.0 | 3,200 | This study | |
| N15 (TF) 3′ | TCAAAATACCCCCGTAAT | 43.0 | This study | |||||
| (CP)EcS1650, | (CP)EcS1650 (TF) 5′ | ATGGCAGTAAAGATTTCA | TFcp | 52.3 | 55.8 | 2,916 | This study | |
| CP933M, λ (TF) 5 | (CP)EcS1650 (TF) 3′ | TTATGCAAGCCTCACAAT | 56.6 | This study | ||||
| T7 (TF) | T7 (TF) 5′ | ATGGCTTAACGTAATTAA | TFT7 | 50.0 | 48.0 | 1,662 | This study | |
| T7 (TF) 3′ | TTACACGTCCTCTACGGC | 58.0 | This study | |||||
| HK022, | HK022 (TF) 5′ | TTGCCTGGAGAAAATATG | TF022 | 56.0 | 57.7 | 1,116 | This study | |
| HK022 (TF) 3′ | TTAGTCAACAAGCTCCCT | 54.6 | This study | |||||
| HK97 (31111) (TF) | HK97 (31111)(TF) 5′ | ATGATTTATAGCACCGGA | TF97 | 55.3 | 58.3 | 795 | This study | |
| HK97 (31111)(TF) 3′ | TTATACTGCCCTTACAATGTA | 54.0 | This study | |||||
| Terminase | 933W Major | 933W Mterm 5′ | ATGACATTCCGGAAGAAT | Term1 | 56.7 | 51.0 | 1,272 | This study |
| Terminase | 933W MTerm 3′ | TCAGTGAGCCATGCAGTG | 62.5 | This study | ||||
| 933W Minor | 933W 5′ MinTerm | ATGGCAAAGCTGGACTGG | Term2 | 63.9 | 64.9 | 807 | This study | |
| Terminase | 933W MinTerm 3′ | TCATTCTTCCGGAATGTCAT | 61.9 | This study |
a
Phage Accession numbers: BP 4795, NC_004813; 933W, NC_000924; partial sequence H19B, AF034975; Stx II bacteriophage I, NC_003525; HK97, NC_002167; Lambda, NC_001416; Nil 2, AJ413274; Stx II bacteriophage II, NC_004914; VT2-Sa, NC_000902; HK620, NC_002730; D3112, NC_005178; P27, NC_003356; phi 105, NC_004167; ST64T, NC_004348; Sakai, NC_002695; P22, NC_002371; ST104, NC_005841; SfV, NC_003444; N15, NC_001901; EcS1650, NC_002695. All CP933 sequences are from EDL 933, NC_002655; T7, NC_001604; HK022, NC_002166; Phi 80, X04051.
b
Tm, thermal denaturation midpoint temperature. Data are presented only for the primers designed for this study.