TABLE 2.
Sequences and concentrations of the primers and cycling conditions used in PCR assays
| PCR | Cycling conditions | Target | Primer | Nucleotide sequence (5′ → 3′) | Amplimer size (bp) | Accession no. on which primer sequence is based | Amount (pmol/PCR) |
|---|---|---|---|---|---|---|---|
| wzz-PCR specific for Y. pestis and Y. pseudotuberculosis | 1 cycle of initial denaturation at 94°C for 2 min; 40 | wzz | Ypf-20120 | GGTGATGAGCAAGTTCAAG | 418 | AJ251713 | 25 |
| cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s; 1 | Ypr-20538 | GCTAAATCCACTGCTCGCTG | AJ251713 | 25 | |||
| cycle of final extension at 72°C for 6 min | |||||||
| wzz-wbyJ-PCR specific for Y. pestis | 1 cycle of initial denaturation at 94°C for 2 min; 35 | wzx-wbyJ | Ypf-8608 | GAAATGGAGAAAAAATCCACGG | 1,612 | AJ251713 | 25 |
| cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s; 1 | Ypr-10219 | GTAGTTATAATACGGTTTACTTAACAACTC | AJ251713 | 25 | |||
| cycle of final extension at 72°C for 6 min | |||||||
| Multiplex PCR | 1 cycle of initial denaturation at 94°C for 2 min; 35 | gmd-fcl | Ypf-14159 | TCAAGATCGCCATGAGAC | 1,370 | AJ251713 | 10 |
| cycles of denaturation at 94°C for 15 s, annealing at 53°C for 30 s, extension at 72°C for 90 s; 1 cycle of final extension at 72°C for 5 min | Ypr-15549 | AGGTTCATTCGTTGGTTC | AJ251713 | 10 | |||
| ddhC-prt | Ypf-5270 Ypr-6342 | CGCATAGAAGAGTTTGTTG CTTTCGCCTGAAATTAGAC | 1,072 | AJ251713AJ251713 | 12.5 12.5 | ||
| manB | Ypf-18740 | GCGAGCCATAACCCAATAGAC | 963 | AJ251713 | 3 | ||
| Ypr-19703 | GCCACCCATCAAATTCCATAC | AJ251713 | 3 | ||||
| abe | Abe1 | AGAATAGTTCTGACTGGAGGAAG | 775 | L01777 | 7.5 | ||
| Abe2 | TCAGGAGCCATTACCTCATC | L01777 | 7.5 | ||||
| wbyL | Ypf-17770 | TTGGAGAAACAAACCTATCTGG | 644 | AJ251713 | 7.5 | ||
| Ypr-18414 | TTTGCATAAAAACGACATAGGC | AJ251713 | 7.5 | ||||
| wbyH | Ypf-7170 | CGTTATCCCAAAAAAGAGG | 528 | AJ251713 | 5 | ||
| Ypr-7698 | ATGGGAGACGCTTGTGATG | AJ251713 | 5 | ||||
| ddhA-B | Ypf-3057 | TGTCGCCTAAAGTTATCG | 407 | AJ251713 | 12.5 | ||
| Ypr-3464 | CGAATATCACCGATTTCC | AJ251713 | 12.5 | ||||
| wbyK | Ypf-13231 | CCGATTACCAGATTTTGAC | 307 | AJ251713 | 7.5 | ||
| Ypr-13538 | CAAAATTCTTATAACCACCACG | AJ251713 | 7.5 | ||||
| wzx | Ypf-8576 | GAAATTCGCATGTAAAAGCTATTG | 105 | AJ251713 | 10 | ||
| Ypr-8681 | GAACCTAGACTTACCACCCCCAAC | AJ251713 | 10 | ||||
| Additional double PCRs | 1 cycle of initial denaturation at 94°C for 2 min; | wzz-gsk | Ypf-20511 | GAAAAATACAGCGAGCAG | 742 | AJ251713 | 15 |
| 35 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 60 s; 1 cycle of final extension at 72°C for 5 min | Yerfb2 | GAYTTGCGYTTACCAGGAAATTTCATTG | Z47767, U13685, U46859 | 15 | |||
| hemH-ddhD | Ypf-913 | CAATCCAATGAAGAGTCAG | 181 | AJ251713 | 15 | ||
| Ypr-1094 | CCCTATGACATAAAAACCC | AJ251713 | 15 | ||||
| Long-range PCR | 1 cycle of initial denaturation at 94°C for 2 min; 10 cycles of denaturation at 92°C for 15 s, annealing at 62°C for 15 s, extension at 68°C for 18 min; 20 cycles of denaturation at 92°C for 15 s, annealing at 62°C for 15 s, extension at 68°C for 18 min + 20 s/cycle; 1 cycle of final extension at 68°C for 7 min | hemH-gsk | yerfb1 yerfb2 | TGGAAGAAATMAAAGARCAAAATCGAGAG GAYTTGCGYTTACCAGGAAATTTCATTG | 20,000 | Z47767, L01777, U46859 Z47767, U13685, U46859 | 25 25 |