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Bacteriology

Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates

Rekha Pai, Robert E. Gertz, Bernard Beall
Rekha Pai
Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
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Robert E. Gertz
Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
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Bernard Beall
Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
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  • For correspondence: BBEALL@CDC.GOV
DOI: 10.1128/JCM.44.1.124-131.2006
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  • FIG. 1.
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    FIG. 1.

    Schematic approach to molecular capsular typing, indicating the seven reactions and the serotypes detected in each reaction.

  • FIG. 2.
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    FIG. 2.

    Representative multiplex reactions. The serotypes of four strains tested for each of eight different multiplex formulations are shown below their respective lanes.

Tables

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  • TABLE 1.

    Oligonucleotide primers used in this study

    Primer pairSanger's strain no./ GenBank accession no.bPrimer sequence (5′→3′)Nucleotide positioncProduct size (bp)
    1-f Z83335 CTC TAT AGA ATG GAG TAT ATA AAC TAT GGT TA10469280
    1-rCCA AAG AAA ATA CTA ACA TTA TCA CAA TAT TGG C10715
    3-f Z47210 ATG GTG TGA TTT CTC CTA GAT TGG AAA GTA G9179371
    3-rCTT CTC CAA TTG CTT ACC AAG TGC AAT AAC G9519
    4-fa AF316639 CTG TTA CTT GTT CTG GAC TCT CGA TAA TTG G9558430
    4-rGCC CAC TCC TGT TAA AAT CCT ACC CGC ATT G9957
    5-f AY336008 ATA CCT ACA CAA CTT CTG ATT ATG CCT TTG TG6123362
    5-rGCT CGA TAA ACA TAA TCA ATA TTT GAA AAA GTA TG6450
    6A/B-f (biotin)d AF316640 AAT TTG TAT TTT ATT CAT GCC TAT ATC TGG8656250
    6A/B-rTTA GCG GAG ATA ATT TAA AAT GAT GAC TA8877
    7F-f554/62CCT ACG GGA GGA TAT AAA ATT ATT TTT GAG13356826
    7F-rCAA ATA CAC CAC TAT AGG CTG TTG AGA CTA AC14150
    7C-fSutcliffCTA TCT CAG TCA TCT ATT GTT AAA GTT TAC GAC GGG A9438260
    7C-rGAA CAT AGA TGT TGA GAC ATC TTT TGT AAT TTC9665
    8-f573/62GAT GCC ATG AAT CAA GCA GTG GCT ATA AAT C8084294
    8-rATC CTC GTG TAT AAT TTC AGG TAT GCC ACC8348
    9V-f980/68CTT CGT TAG TTA AAA TTC TAA ATT TTT CTA AG12345753
    9V-rGTC CCA ATA CCA GTC CTT GCA ACA CAA G13070
    10A-f1006/38GGT GTA GAT TTA CCA TTA GTG TCG GCA GAC12423628
    10A-rGAA TTT CTT CTT TAA GAT TCG GAT ATT TCT C13021
    11A-f1813/39GGA CAT GTT CAG GTG ATT TCC CAA TAT AGT G11640463
    11A-rGAT TAT GAG TGT AAT TTA TTC CAA CTT CTC CC12071
    12F-f6312GCA ACA AAC GGC GTG AAA GTA GTT G14407376
    12F-rCAA GAT GAA TAT CAC TAC CAA TAA CAA AAC14753
    14-f34359CTT GGC GCA GGT GTC AGA ATT CCC TCT AC7968208
    14-rGCC AAA ATA CTG ACA AAG CTA GAA TAT AGC C8145
    15A-f389/39ATT AGT ACA GCT GCT GGA ATA TCT CTT C7114436
    15A-rGAT CTA GTG AAC GTA CTA TTC CAA AC8313
    15B/C-f7904/39TTG GAA TTT TTT AAT TAG TGG CTT ACC TA7314496
    15B/C-rCAT CCG CTT ATT AAT TGA AGT AAT CTG AAC C7779
    16F-fNr.34361CTG TTC AGA TAG GCC ATT TAC AGC TTT AAA TC11521988
    16F-rCAT TCC TTT TGT ATA TAG TGC TAG TTC ATC C12478
    17F-fRose (am)TTC GTG ATG ATA ATT CCA ATG ATC AAA CAA GAG10484693
    17F-rvac.GAT GTA ACA AAT TTG TAG CGA CTA AGG TCT GC11145
    Sg18-f4593/40CTT AAT AGC TCT CAT TAT TCT TTT TTT AAG CC12687573
    Sg18-rTTA TCT GTA AAC CAT ATC AGC ATC TGA AAC13230
    19A-fa AF094575 GTT AGT CCT GTT TTA GAT TTA TTT GGT GAT GT12118478
    19A-rGAG CAG TCA ATA AGA TGA GAC GAT AGT TAG12566
    19F-f U09239 GTT AAG ATT GCT GAT CGA TTA ATT GAT ATC C7828304
    19F-rGTA ATA TGT CTT TAG GGC GTT TAT GGC GAT AG8100
    20-f34365 (am)GAG CAA GAG TTT TTC ACC TGA CAG CGA GAA G9567514
    20-rCTA AAT TCC TGT AAT TTA GCT AAA ACT CTT ATC10048
    22F-f1772/40 (da)GAG TAT AGC CAG ATT ATG GCA GTT TTA TTG TC11055643
    22F-rCTC CAG CAC TTG CGC TGG AAA CAA CAG ACA AC11666
    23F-faAF057294GTA ACA GTT GCT GTA GAG GGA ATT GGC TTT TC12118384
    23F-rCAC AAC ACC TAA CAC ACG ATG GCT ATA TGA TTC12566
    31-fNr.34374 (am)GGA AGT TTT CAA GGA TAT GAT AGT GGT GGT GC9144701
    31-rCCG AAT AAT ATA TTC AAT ATA TTC CTA CTC9815
    33F-f AY163221 GAA GGC AAT CAA TGT GAT TGT GTC GCG181338
    33F-rCTT CAA AAT GAA GAT TAT AGT ACC CTT CTA C488
    34-f676/74GCT TTT GTA AGA GGA GAT TAT TTT CAC CCA AC7350408
    34-rCAA TCC GAC TAA GTC TTC AGT AAA AAA CTT TAC7725
    35B-f4356/39 (da)GAT AAG TCT GTT GTG GAG ACT TAA AAA GAA TG10556677
    35B-rCTT TCC AGA TAA TTA CAG GTA TTC CTG AAG CAA G11199
    35F-f361/39 (da)GAA CAT AGT CGC TAT TGT ATT TTA TTT AAA GCA A7374517
    35F-rGAC TAG GAG CAT TAT TCC TAG AGC GAG TAA ACC7858
    38-f9687/39CGT TCT TTT ATC TCA CTG TAT AGT ATC TTT ATG13848574
    38-rATG TTT GAA TTA AAG CTA ACG TAA CAA TCC14392
    cpsA-f AF057294 GCA GTA CAG CAG TTT GTT GGA CTG ACC2314160
    cpsA-rGAA TAT TTT CAT TAT CAG TCC CAG TC2473
    • ↵ a Primers previously published as in reference 3.

    • ↵ b Primers were designed using the sequence either from the GenBank (accession numbers provided) or from www.sangers.ac.uk (strain numbers provided).

    • ↵ c Start position of each primer.

    • ↵ d The biotinylated forward primer is required for subsequent resolution of serotypes 6A and 6B by pyrosequencing.

  • TABLE 2.

    Primer concentrations in seven multiplex PCRa

    ReactionPrimersPrimer concn (μM)
    119A-f, 19A-r1
    3-f, 3-r1.5
    22F-f, 22-r1.5
    6A/B-f (biotin), 6A/B-r0.5
    24-f, 4-r1.5
    14-f, 14-r1.0
    12F-f, 12F-r1.5
    9V-f, 9V-r1.5
    323F-f, 23F-r1.5
    7F-f, 7F-r2.0
    11A-f, 11A-r1.0
    33F-f, 33F-r1.0
    419F-f, 19F-r1.5
    16F-f, 16F-r2.0
    sg18-f, sg18-r1.25
    35B-f, 35B-r1.0
    58-f, 8-r1.5
    15B/C-f, 15B/C-r1.5
    38-f, 38-r1.5
    31-f, 31-r2.0
    61-f, 1-r1.5
    10A-f, 10-r1.5
    35F-f, 35F-r1.5
    34-f, 34-r1.5
    720-f, 20-r1.5
    7C-f, 7C-r1.5
    17F-f, 17F-r1.5
    15A-f, 15A-r1.5
    • ↵ a The universal capsular primers cpsA-f and cpsA-r were included in all reactions at 0.5 μM each.

  • TABLE 3.

    Serotyping results deduced by sequential multiplex PCR for 421 randomly chosen invasive pneumococcal isolates

    Serotype result(s) from multiplex PCRaActual conventional serotyping result(s) (no. of positive isolates)% of total (n = 421)
    11 (13)3.1
    33 (24)5.7
    44 (23)5.5
    6A/6B, 35 isolates6A (19), 6B (16)8.3
    7F/(7A)7F (20), 7A (2)5.2
    7C/(7B)/(40)7C (8), 7B (1)2.1
    88 (8)1.9
    9V/(9A)9V (15)3.6
    1414 (20)4.8
    2020 (6)1.4
    3131 (8)1.9
    3434 (7)1.6
    38/(25F)38 (8)1.9
    10A10A (9)2.1
    11A/(11D)11A (10)2.3
    12F/(12A)/(44)/(46)12F (21)4.9
    15A15A (9)2.1
    15B/C15B/C (15)3.2
    16F16F (9)2.1
    17F17F (10)2.3
    18C/(18B)/(18F)/(18A), 3 isolates18C (8), 18B (3), 18F (1), 18A (1)3.1
    19A19A (34)8.1
    19F19F (10)2.3
    22F/(22A)22F (19), 22A (1)5.0
    23F23F (16)3.8
    33F/(33A)/(37)33F (10)2.4
    35B35B (8)1.9
    35F/(47F)35F (9)2.1
    Nontypeable9N (4), 10F (1), 13 (2), 21 (2), 23A (1), 23B (3), 28A (1), 28F (1), 29 (1), 41A (1), NT (3)4.8
    • ↵ a Serotypes in parentheses occur in 0 to 0.2% of ABCs isolates (according to year 2003 data).

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Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates
Rekha Pai, Robert E. Gertz, Bernard Beall
Journal of Clinical Microbiology Jan 2006, 44 (1) 124-131; DOI: 10.1128/JCM.44.1.124-131.2006

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Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates
Rekha Pai, Robert E. Gertz, Bernard Beall
Journal of Clinical Microbiology Jan 2006, 44 (1) 124-131; DOI: 10.1128/JCM.44.1.124-131.2006
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KEYWORDS

Bacterial Capsules
Bacterial Typing Techniques
polymerase chain reaction
Streptococcus pneumoniae

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