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. 2014 May;52(5):1714–1719. doi: 10.1128/JCM.03411-13

TABLE 1.

Primer sequences used in this study and the predicted sizes of the PCR products

Target cps group or type Primer sequences (5′ to 3′)
 Target gene(s)a Putative gene product(s) (HGb) Size (bp) of products
Forward Reverse
For grouping PCRc
    I TGGTTCAAATATCAATGCTC ATTGGTTGTGAGTGCATTG cps3G, cps13G, cps18G Aminotransferase (HG41) 933
    IId TCAAAATACGCACCTAAGGC CACTCACCTGCCCCAAGAC cps1Q, cps1/2P, cps2P, cps6Q, cps14P, cps16R, cps27P N-acetylneuraminic acid synthase NeuB (HG10) 823
    IIIe,f TGATTTGGGTGAGACCATG CTCATGCTGGATAACACGT cps21R, cps28P, cps29Q, cps30N N-acetylfucosamine synthase FlnC (HG26) 583
    IV ACAGTCGGTCAAGATAATCG TCAGCTTGGGTAATATCTGG cps4F-G, cps5F-G, cps7F-G, cps17G-H, cps19G-H, cps23F-G Initial sugar transferase (HG21) and aminotransferase (HG22) 455
    Vg GGAAAGATGGAGGACCAGC CCAACCAGACTCATATCCCC cps8E, cps15E, cps20E, cps22E, cps25E Initial sugar transferase (HG6) 265
    III and VIe,h GATGCCCCAAGCGATATGCC (F1), GACGCACCAAGTGATATGCC (F2) GGACCAACAATGGCCATCTC (R1), GGTCCGACAATAGCCATTTC (R2) Group III: cps21G, cps28F, cps29G, cps30F; group VI, cps9F, cps10F, cps11F, cps12F, cps24G, cps26H, cps33G Initial sugar transferase (HG8) 146
For typing PCR
    cps group I
        3 GGTTTTGATTGGTCTAGTTG CTCTAAAGCTCGATATCTAC cps3L Wzy polymerase (HG90) 214
        13 TATGGTTAAAGGTGGAACTG CCTTGTATATATTCCCTCCA cps13L Wzy polymerase (HG148) 408
        18 TAATGGGATAGTTGCGTTAC ATACATAAAGTTGTCCTGCG cps18N Wzy polymerase (HG170) 617
    cps group II
        2 and 1/2 TTAGCAACGTTGCCAATAAG AATCCTCCATTAAAACCCTG cps2I, cps1/2I Wzy polymerase (HG54) 173
        6 GCTCACTATTTTTACATTACAC TATTACTCCGCCAAATACAG cps6I Wzy polymerase (HG109) 278
        1 and 14 TTAGACAGACACCTTATAGG CTAGCTTCGTTACTTGATTC cps14H, cps1I Wzy polymerase (HG50) 386
        16 AAGGTTATCCACGAAAGATG TCCGGCAATATTCTTTCAAG cps16I Wzy polymerase (HG362) 494
        27 AGACACTGCTTGCATTATTG TCAGAATTACTTCCTGTTGC cps27K Wzy polymerase (HG246) 655
    cps group III
        21 TATCATATTGAGAATCTTCCC TTGCGTAGCATACAAAGTTC cps21P Wzy polymerase (HG194) 160
        28 ATTATGTTGGTTGCAGAAGG CGACTCAATTGTTGTAGTAG cps28L Wzy polymerase (HG254) 272
        29 TTCTGGGATTTTAGGAATGC CATGAAATACGCACTTGTAC cps29L Wzy polymerase (HG259) 415
        30 TATTGCACTAGCTTCAGAAC TGCATCCATAGTTGTATTCG cps30I Wzy polymerase (HG264) 568
    cps group IV
        4 GACTATCTGTATACCCAAAC TCCTTCCAAGTATTCTCTAG cps4K Wzy polymerase (HG96) 903
        5 ATCTTAGGAATGATTCGGAC ACCAGATATCTGAGCAAATG cps5L Wzy polymerase (HG103) 720
        7 AACTACCTACCTGAACTTTG AGTCTAAAAGTGATCGAGTC cps7L Wzy polymerase (HG113) 566
        17 TAGCATCAGTTTATACGAGG TAGTTTATCTGTGACACACC cps17O Wzy polymerase (HG164) 455
        19 GTGTCGCAAATCAAGTATTG AAGCTAGTACAACAAGCATG cps19L Wzy polymerase (HG174) 348
        23 TAATGTATGCTCTGTCACTG AACGAAACGGAATAGTTTGC cps23J Wzy polymerase (HG213) 221
    cps group V
        8 AAATAAGGTAGGAGCTACTC ATCCAACCTTAGCTTTCTGT cps8K Wzy polymerase (HG120) 446
        15 ATCGTTTTGAGATTGAGTGG TAAACGGATTCGGTTACTCA cps15K Wzy polymerase (HG153) 542
        20 TGTGGATTTCTGGGATAATC TGTGGACGAATTACTACTTG cps20I Wzy polymerase (HG182) 698
        22 GCATTATCAGGATTCTTTCC CCAATTGGGTGTTCAAAAAG cps22K Wzy polymerase (HG200) 296
        25 GTTTGCTCCGATCATAATAG CCAGTAAAAGGACTCAATAC cps25M Wzy polymerase (HG229) 174
    cps group VI
        9 GAAAGTAGGTATATCTCAGC GGGCTATTAAAACTCCTATC cps9J Wzy polymerase (HG123) 368
        10 TTTCCCATTTGCTTATGGAC GGAATAAAAACGATTGGGAG cps10M Wzy polymerase (HG130) 633
        11 ATGCGATTGCAACAATTGAC AGGCATGAGTAATACATAGG cps11N Wzy polymerase (HG138) 833
        12 AACAGGTATTTCAGGATTGC CTCGGATAAAGATAATCAGC cps12J Wzy polymerase (HG140) 131
        24 TACTGAGATTTATTGGGACG AAGCGATTGGATTACATTGC cps24M Wzy polymerase (HG220) 224
        26 TTATACCGAAATTTTGTTGCC CGTCAATCATATAAAGTGGG cps26P Wzy polymerase (HG240) 472
        33 GATGTTTTCAACAGGTGTAC CAAAGTACCTATTTTCAGCG cps33K Wzy polymerase (HG286) 710
    cps group VII
        31 ACAATCGTTTCTGCAATACG GATGAAAACATCGTTGGTAG cps31L Wzy polymerase (HG274) 842
ATCAGTAGTGGGAATAGTTG TTTACTGTTTTTCGACCGTG cps31G Initial sugar transferase (HG269) 423
        32 AACCGCTGTTGAATTAAGAG TTCGTTAGTTGAACTGTTCC cps32H Wzy polymerase (HG281) 570
TAGGACTATGGTTCCTAATG TATTCTAGTTCAAGTCGCTC cps32E Initial sugar transferase (HG278) 342
        34 AAGTTTCATTCGAGGACTTC GTATATAACACCGCAAGAAG cps34M Wzy polymerase (HG287) 246
ATACAGTGATGTCTTGCAAC ATTGCTTTTTGACAATCGGC cps34E Initial sugar transferase (HG290) 701
For all PCR
    Internal control GAGTTTGATCCTGGCTCAG AGAAAGGAGGTGATCCAGCC 16S rRNA 1,542–1,553
a

Refer to our previous study (8) for gene names.

b

HG, homology group of putative cps gene products assigned in our previous study (8).

c

Only the internal control products were amplified from cps group VII strains.

d

Serotype 13 reference strain has a homologue of target genes for cps group II (putative N-acetylneuraminic acid synthase gene, cps13O). However, the primers were designed to not detect this gene.

e

In addition to the internal control products, cps group III strains produced two cps gene products by the grouping PCR (146- and 583-bp products), and the 146-bp product is common with cps group VI strains.

f

Serotype 17 reference strain has a homologue of target genes for cps group III (putative N-acetylfucosamine synthase gene, cps17R). However, the primers were designed to not detect this gene.

g

All serotype reference strains in cps-group II also possess the homologues of target genes for cps group V (genes encoding putative initial sugar transferase of HG6). However, the primers were designed not to detect these strains.

h

Because the genes encoding putative initial sugar transferase of HG8 have sequence variations among serotype reference strains in groups III and VI, two primers were designed for each primer site to amplify the 146-bp products. Of note, all reference strains in groups III and VI were detected only when the four primers were mixed, while with every single primer pair (forward 1 [F1]-reverse 1 [R1], F1-R2, F2-R1, or F2-R2), some of the reference strains in groups III and VI were not detected.