TABLE 2.
Primers and thermal cycler programs used for the PCRs in this study
| Probe | Primer | Primer sequence (5′-3′) | Primer 5′ positiona | Programb |
|---|---|---|---|---|
| 1 | Tn4R | GTCCTGTGGCAATGAAGACAATGC | 51 ORF 1 | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min |
| Tn15 | TCCTCCATGTAATATTCCACCGCC | 171 ORF 3 | ||
| 2 | Tn15R | GGCGGTGGAATATTACATGGAGGA | 148 ORF 3 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn15A | GCTGCTACTGGAGCGAGAGTAAGT | 363 ORF 5 | ||
| 3 | Tn15AR | ACTTACTCTCGCTCCAGTAGCAGC | 387 ORF 5 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn24R | CGGTAGTATTCACATAGCTCGCC | 75 ORF 7 | ||
| 4 | Tn24 | GGCGAGCTATGTGAATACTACCG | 97 ORF 7 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn25 | CGCAGGTCAGACCCTGAGTCGA | 823 ORF 9 | ||
| 5 | Tn1 | GGTGTGCCATCGCAATGAAGCA | 1,169 ORF 14 (rteA) | 94°C, 1 min; 55°C, 1 min; 66°C, 2 min |
| Tn2 | CTCGGTATGGAGTATGCTCCAG | 111 ORF 16 | ||
| 6 | 15 | GGAACTTCGTGCTGCCCTCAAAG | 511 ORF 25 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn7 | GTAATTCGGTATCGTCCACCAGC | 628 ORF 27 | ||
| 7 | Tn11 | CAGCTACTCATGCCGTTCCAGGC | 242 ORF 28 | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min |
| Tn12 | CGTTTGGCTCATTGTTCGGGCTG | 624 ORF 29 | ||
| 8 | Tn13 | CCACCGGTTGCTCCTTCGTTTC | 1,060 ORF 30 | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min |
| Tn14 | GTGCAACCGTTGTCGCACGTAC | 101 ORF 30 | ||
| 9 | TranF | CGCAGAAGATCGAGGAACTCAC | 62 traN (CTn9343) | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| TranR | CGCTGTCAGCAGCGACTTGTTC | 1,056 traN (CTn9343) | ||
| 10 | Tn18 | CGAACAGCATGTCCGTGTTGCC | 2,125 ORF 42 (traG) | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min |
| Tn19 | AGCCTTCAGGCACATGCCCGAC | 171 ORF 42 (traG) | ||
| 11 | Tn20 | GCGCGTCCCGATTGTTTCCAGG | 615 ORF 55 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn21 | GGGGATACGACATGTACCGCTC | 86 ORF 56 | ||
| 12 | Tn26 | CCGCAGAACGGCATATACCCTGG | 201 ORF 57 | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min |
| Tn27 | CATGGGTGCGAGGTCCGTCCCTC | 127 ORF 59 | ||
| 13 | Tn22 | CGTGGACACGACTGCTGGAAGT | 259 ORF 61 | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min |
| Tn23 | GGGGCACTTCTTCAACGATGGG | 198 ORF 63 | ||
| 14 | Tn5B | CGGTTGTACAGCTCCTCTTCTGTC | 728 ORF 65 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn5AR | GCTGCATGACATCGGTCGTATGAC | 1,244 ORF 66 | ||
| 15 | Tn5A | GTCATACGACCGATGTCATGCAGC | 1,267 ORF 66 | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min |
| Tn5R | CTCTGTGAATAAGGTGGCTTGCC | 568 upstream ORF 66 | ||
| Tn2 | CTCGGTATGGAGTATGCTCCAG | 111 ORF 16 (bexA) | 94°C, 1 min; 55°C, 1 min; 66°C, 2 min | |
| Tn2A | GTATTCCACTGCCCAATATGCCGC | 625 ORF 16 | ||
| 86CTn8 | GGTTCTGGCAGGAGGAGTTTACC | 1,086 dprA (NCTC 9343) | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min | |
| 86CTn2 | GTATCGTACAATAGAGAGGTCGCC | 1,249 tnpA1 (CTn86) | ||
| Tn22 | CGTGGACACGACTGCTGGAAGT | 259 tnpA2 (CTn86) | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min | |
| 86CTn9 | CCAGCAACGACCTTCAACGCCTC | 693 ORF-C (NCTC 9343) | ||
| 86CTn2 | GTATCGTACAATAGAGAGGTCGCC | 1,249 tnpA1 (CTn86) | 94°C, 1 min; 51°C, 1 min; 72°C, 2 min | |
| Tn22 | CGTGGACACGACTGCTGGAAGT | 259 tnpA2 (CTn86) | ||
| Tn25A | CCTGTACGACTATGACAGGGCG | 240 tnpB (CTn9343) | 94°C, 1 min; 51°C, 1 min; 72°C, 2 min | |
| Tn22 | CGTGGACACGACTGCTGGAAGT | 259 tnpA2 (CTn9343) | ||
| Tn24 | GGCGAGCTATGTGAATACTACCG | 97 ORF-7 (NCTC 9343) | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min | |
| Tn23 | GGGGCACTTCTTCAACGATGGG | 198 ORF-63 (NCTC 9343) | ||
| E11.2 | TGTGCGGGAACTCCTCTTGCTCTTCCTT | 257 upstream bfmA (CTn86) | 94°C, 1 min; 66°C, 30 s; 68°C, 4 min | |
| E13 | GCCGGATCGTGAAAAGGAATCG | 243 bfmA (CTn86) | ||
| 86CTn2 | GTATCGTACAATAGAGAGGTCGCC | 1,249 tnpA1 (CTn86) | 94°C, 60 s; 55°C, 60 s; 66°C, 2 min | |
| 86CTn1R | GCCTTCTGTGTCCTTCACCATAGA | 1,201 tnpA1 (CTn86) | ||
| Tn25D | GGGCAACTGTCCGTCCTGTCTG | 1,583 tnpA1 (CTn9343) | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min | |
| Tn25 | CGCAGGTCAGACCCTGAGTCGA | 823 tnpA1 (CTn9343) | ||
| Tn25E | ATGTATCTACGGACCGACAGGC | 133 int2 (CTn9343) | 94°C, 1 min; 62°C, 1 min; 68°C, 2 min | |
| Tn25C | ATGTCTCCTCACATGCCACGTC | 669 int2 (CTn9343) | ||
| Tn1 | GGTGTGCCATCGCAATGAAGCA | 1,169 rteA (CTn9343) | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min | |
| Tn1A | GGCGAGACTGTATTGGAGTGGG | 190 rteA (CTn9343) | ||
| Tn21A | CAGGCGTTCCGCCTCGTTCACC | 840 prmN1 (CTn9343) | 94°C, 1 min; 66°C, 1 min; 70°C, 2 min | |
| Tn21 | GGGGATACGACATGTACCGCTC | 86 prmN1 (CTn9343) |
a
In base pairs (primers used to construct the DNA probes were derived from CTn9343 and its flanking element).
b
All programs had an initial segment at 94°C for 1 min (except PCR with primers 86CTn2 and Tn22 and Tn25A and Tn22, which had an initial segment at 94°C for 5 min), were run for 30 cycles and had a 10-min final extension at the corresponding extension temperature. The programs to identify CTn86 and CTn9343 circular intermediate form (primers 86CTn2 and Tn22 and Tn25A and Tn22) were run for 20, 25, and 30 cycles to detect the PCR product in the linear range of the reaction.