Internal fragments of the 5-enolpyruvylshikimate 3-phosphate synthase (aroA), 60 KDa chaperonin (cpn60), peroxide resistance (dpr), glucose kinase (gki), DNA mismatch repair protein (mutS), homologous recombination factor (recA) and aspartokinase (thrA) genes were amplified by PCR using the following primer pairs.
aroA-up 5’ TTCCATGTGCTTGAGTCGCTA 3’ and aroA-dn 5’ ACGTGACCTACCTCCGTTGAC 3’ cpn-up 5’ TTGAAAAACGTRACKGCAGGTGC 3’and cpn-dn 5’ ACGTTGAAIGTACCACGAATC 3’ dpr-up 5’ CGTCTTTCAGCCCGCGTCCA 3’ and dpr-dn 5’ GACCAAGTTCTGCCTGCAGC 3’ gki-up 5’ GGAGCCTATAACCTCAACTGG 3’ and gki-dn 5’ AAGAACGATGTAGGCAGGATT 3’ mutS-up 5’ CGCAGAGCAGATGGAAGATCC 3’ and mutS-dn 5’ CCCATAGCTGTTTTGGTTTCATC 3’ recA-up 5’ TATGATGAGTCAGGCCATG 3’ and recA-dn 5’ CGCTTAGCATTTTCAGAACC 3’ thrA-up 5’ GATTCAGAACGTCGCTTTGT 3’ and thrA-dn 5’ AAGTTTTCATAGAGGTCAGC 3’
The primer
5’ AAGAATGGATCATCAACCGT 3’
was used for the forward thrA sequencing reaction.
PCR reactions are performed in 50 µl volumes using 30 cycles of 95 °C for 1 min, X °C for 1 min. (where X is 55 °C for aroA and gki, 52 °C for cpn and thrA, and 50 °C for dpr, mutS and recA) and 72 °C for 1 min. PCR products are purified through Qiagen PCR purification kits and the sequence of each fragment is obtained on both strands using the same primers as those used in the initial PCR with the exception of thrA as described above.
The development of the initial Streptococcus suis MLST database is described in the following publication:
King, S.J., Leigh, J.A., Heath, P.J., Luque, I., Tarradas, C., Dowson, C.G., and Whatmore, A.M. 2002. Development of a multilocus sequence typing scheme for Streptococcus suis: Identification of virulent clones and potential capsular serotype exchange. J Clin Microbiol 40:3671-3680.
The Streptococcus suis MLST database was developed by Sam King and Adrian Whatmore at the University of Warwick. We gratefully acknowledge the many co-workers who contributed strains for the pilot study (A. Cheng, B. Francois, M. Gottschalk, G. Grise, P. Heath, C. Lammler, I. Luque and P. Norton) and the financial support of the BBSRC (Grant 88/S11598) and The Wellcome Trust (Grant 053589).