PCR for Staphylococcus epidermidis MLST

Internal fragments of the seven loci can be amplified by PCR, using the primers listed below and chromosomal DNA as a template. PCR involved an initial denaturation of 95 °C for 3 min; 34 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min; and a final extension of 72 °C for 10 min.

Gene and function Primer Sequences (5'-3') Size of amplicon used for assigning alleles
Carbamate Kinase (arcC) arcC-F TGTGATGAGCACGCTACCGTTAG 465
arcC-R TCCAAGTAAACCCATCGGTCTG
Shikimate dehydrogenase (aroE) aroE-F CATTGGATTACCTCTTTGTTCAGC 420
aroE-R CAAGCGAAATCTGTTGGGG
ABC transporter (gtr) gtr-F CAGCCAATTCTTTTATGACTTTT 438
gtr-R GTGATTAAAGGTATTGATTTGAAT
DNA mismatch repair protein (mutS) mutS-F3 GATATAAGAATAAGGGTTGTGAA 412
mutS-R3 GTAATCGTCTCAGTTATCATGTT
Pyrimidine operon regulatory protein (pyrR) pyr-F2 GTTACTAATACTTTTGCTGTGTTT 428
pyr-R4 GTAGAATGTAAAGAGACTAAAATGAA
Triosephosphate isomerase (tpiA) tpi-F2 ATCCAATTAGACGCTTTAGTAAC 424
tpi-R2 TTAATGATGCGCCACCTACA
Acetyl coenzyme A acetyltransferase (yqiL) yqiL-F2 CACGCATAGTATTAGCTGAAG 416
yqiL-R2 CTAATGCCTTCATCTTGAGAAATAA

Sequences for each locus must be obtained for both the forward and reverse strands, and must be 100% accurate, since even a single error will alter the allelic number obtained.