Table 1 PCR protocol for specific Kingella kingae multilocus sequence typing
Primer design | ||||
Gene | Primers name | Primers | Primer length (bp) | Amplicon length (bp) |
abcZ | abcZ_Kki_Fwd | CGCAAGAAAGCGTGTTTGAC | 20 | 532 |
abcZ_Kki_Rev | CAATTCCTGCGCCTTTTTCTC | 21 | ||
adk | adk_Kki_Fwd | CACACAAGCGCAATTTATTACG | 22 | 491 |
adk_Kki_Rev | AAACTTCGGTTTGTTCGTGATAT | 23 | ||
aroE | aroE_Kki_Fwd | CAAATCCCCACAAATTCATCAATG | 24 | 621 |
aroE_Kki_Rev | AACGCGGTGGGCTGGTTC | 18 | ||
cpn60 | cpn60_Kki_Fwd | CATGGGCGCACAAATGGTT | 19 | 467 |
cpn60_Kki_Rev | CAAACAACAACACAAATGGGC | 21 | ||
recA | recA_Kki_Fwd | GACGGCAGCCACCAAGAC | 21 | 456 |
recA_Kki_Rev | TCCTGCCAGTTTACGCAAG | 19 | ||
gdh/zwf | gdh/zwf_Kki_Fwd | GAGCGCGGCGAGTTTTAT | 18 | 671 |
gdh/ zwf_Kki_Rev | CAGTTGTCCAAAATTGGCATG | 21 | ||
10× PCR Buffer | 1× | |||
25 mM MgCl2 | 2.0 mM | |||
dNTP mix (10 mM of each) | 200 μM of each dNTP | |||
Forward primer | 0,1 μM | |||
Reverse primer | 0,1 μM | |||
HotStarTaq DNA Polymerase | 2.5 units/ reaction | |||
Distilled water | variable | |||
Template DNA | < 0,5 μg | |||
Total volume | 50 μl | |||
PCR protocol | ||||
Cycle step | 3 step-protocol | Cycles | ||
Temperature | Time | |||
Initial denaturation | 95 °C | 15 min | 1 | |
Denaturation | 95 °C | 1 min | 35 | |
Annealing | 58 °C | 30 s | ||
Elongation | 72 °C | 1 min 30 s | ||
Final elongation | 72 °C | 10 min | 1 | |