DOI: 10.1128/JCM.44.1.124-131.2006
Article Figures & Data
Figures
- FIG. 1.
Schematic approach to molecular capsular typing, indicating the seven reactions and the serotypes detected in each reaction.
- 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
- TABLE 1.
Oligonucleotide primers used in this study
Primer pair Sanger's strain no./ GenBank accession no.b Primer sequence (5′→3′) Nucleotide positionc Product size (bp) 1-f Z83335 CTC TAT AGA ATG GAG TAT ATA AAC TAT GGT TA 10469 280 1-r CCA AAG AAA ATA CTA ACA TTA TCA CAA TAT TGG C 10715 3-f Z47210 ATG GTG TGA TTT CTC CTA GAT TGG AAA GTA G 9179 371 3-r CTT CTC CAA TTG CTT ACC AAG TGC AAT AAC G 9519 4-fa AF316639 CTG TTA CTT GTT CTG GAC TCT CGA TAA TTG G 9558 430 4-r GCC CAC TCC TGT TAA AAT CCT ACC CGC ATT G 9957 5-f AY336008 ATA CCT ACA CAA CTT CTG ATT ATG CCT TTG TG 6123 362 5-r GCT CGA TAA ACA TAA TCA ATA TTT GAA AAA GTA TG 6450 6A/B-f (biotin)d AF316640 AAT TTG TAT TTT ATT CAT GCC TAT ATC TGG 8656 250 6A/B-r TTA GCG GAG ATA ATT TAA AAT GAT GAC TA 8877 7F-f 554/62 CCT ACG GGA GGA TAT AAA ATT ATT TTT GAG 13356 826 7F-r CAA ATA CAC CAC TAT AGG CTG TTG AGA CTA AC 14150 7C-f Sutcliff CTA TCT CAG TCA TCT ATT GTT AAA GTT TAC GAC GGG A 9438 260 7C-r GAA CAT AGA TGT TGA GAC ATC TTT TGT AAT TTC 9665 8-f 573/62 GAT GCC ATG AAT CAA GCA GTG GCT ATA AAT C 8084 294 8-r ATC CTC GTG TAT AAT TTC AGG TAT GCC ACC 8348 9V-f 980/68 CTT CGT TAG TTA AAA TTC TAA ATT TTT CTA AG 12345 753 9V-r GTC CCA ATA CCA GTC CTT GCA ACA CAA G 13070 10A-f 1006/38 GGT GTA GAT TTA CCA TTA GTG TCG GCA GAC 12423 628 10A-r GAA TTT CTT CTT TAA GAT TCG GAT ATT TCT C 13021 11A-f 1813/39 GGA CAT GTT CAG GTG ATT TCC CAA TAT AGT G 11640 463 11A-r GAT TAT GAG TGT AAT TTA TTC CAA CTT CTC CC 12071 12F-f 6312 GCA ACA AAC GGC GTG AAA GTA GTT G 14407 376 12F-r CAA GAT GAA TAT CAC TAC CAA TAA CAA AAC 14753 14-f 34359 CTT GGC GCA GGT GTC AGA ATT CCC TCT AC 7968 208 14-r GCC AAA ATA CTG ACA AAG CTA GAA TAT AGC C 8145 15A-f 389/39 ATT AGT ACA GCT GCT GGA ATA TCT CTT C 7114 436 15A-r GAT CTA GTG AAC GTA CTA TTC CAA AC 8313 15B/C-f 7904/39 TTG GAA TTT TTT AAT TAG TGG CTT ACC TA 7314 496 15B/C-r CAT CCG CTT ATT AAT TGA AGT AAT CTG AAC C 7779 16F-f Nr.34361 CTG TTC AGA TAG GCC ATT TAC AGC TTT AAA TC 11521 988 16F-r CAT TCC TTT TGT ATA TAG TGC TAG TTC ATC C 12478 17F-f Rose (am) TTC GTG ATG ATA ATT CCA ATG ATC AAA CAA GAG 10484 693 17F-r vac. GAT GTA ACA AAT TTG TAG CGA CTA AGG TCT GC 11145 Sg18-f 4593/40 CTT AAT AGC TCT CAT TAT TCT TTT TTT AAG CC 12687 573 Sg18-r TTA TCT GTA AAC CAT ATC AGC ATC TGA AAC 13230 19A-fa AF094575 GTT AGT CCT GTT TTA GAT TTA TTT GGT GAT GT 12118 478 19A-r GAG CAG TCA ATA AGA TGA GAC GAT AGT TAG 12566 19F-f U09239 GTT AAG ATT GCT GAT CGA TTA ATT GAT ATC C 7828 304 19F-r GTA ATA TGT CTT TAG GGC GTT TAT GGC GAT AG 8100 20-f 34365 (am) GAG CAA GAG TTT TTC ACC TGA CAG CGA GAA G 9567 514 20-r CTA AAT TCC TGT AAT TTA GCT AAA ACT CTT ATC 10048 22F-f 1772/40 (da) GAG TAT AGC CAG ATT ATG GCA GTT TTA TTG TC 11055 643 22F-r CTC CAG CAC TTG CGC TGG AAA CAA CAG ACA AC 11666 23F-fa AF057294 GTA ACA GTT GCT GTA GAG GGA ATT GGC TTT TC 12118 384 23F-r CAC AAC ACC TAA CAC ACG ATG GCT ATA TGA TTC 12566 31-f Nr.34374 (am) GGA AGT TTT CAA GGA TAT GAT AGT GGT GGT GC 9144 701 31-r CCG AAT AAT ATA TTC AAT ATA TTC CTA CTC 9815 33F-f AY163221 GAA GGC AAT CAA TGT GAT TGT GTC GCG 181 338 33F-r CTT CAA AAT GAA GAT TAT AGT ACC CTT CTA C 488 34-f 676/74 GCT TTT GTA AGA GGA GAT TAT TTT CAC CCA AC 7350 408 34-r CAA TCC GAC TAA GTC TTC AGT AAA AAA CTT TAC 7725 35B-f 4356/39 (da) GAT AAG TCT GTT GTG GAG ACT TAA AAA GAA TG 10556 677 35B-r CTT TCC AGA TAA TTA CAG GTA TTC CTG AAG CAA G 11199 35F-f 361/39 (da) GAA CAT AGT CGC TAT TGT ATT TTA TTT AAA GCA A 7374 517 35F-r GAC TAG GAG CAT TAT TCC TAG AGC GAG TAA ACC 7858 38-f 9687/39 CGT TCT TTT ATC TCA CTG TAT AGT ATC TTT ATG 13848 574 38-r ATG TTT GAA TTA AAG CTA ACG TAA CAA TCC 14392 cpsA-f AF057294 GCA GTA CAG CAG TTT GTT GGA CTG ACC 2314 160 cpsA-r GAA TAT TTT CAT TAT CAG TCC CAG TC 2473 ↵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
Reaction Primers Primer concn (μM) 1 19A-f, 19A-r 1 3-f, 3-r 1.5 22F-f, 22-r 1.5 6A/B-f (biotin), 6A/B-r 0.5 2 4-f, 4-r 1.5 14-f, 14-r 1.0 12F-f, 12F-r 1.5 9V-f, 9V-r 1.5 3 23F-f, 23F-r 1.5 7F-f, 7F-r 2.0 11A-f, 11A-r 1.0 33F-f, 33F-r 1.0 4 19F-f, 19F-r 1.5 16F-f, 16F-r 2.0 sg18-f, sg18-r 1.25 35B-f, 35B-r 1.0 5 8-f, 8-r 1.5 15B/C-f, 15B/C-r 1.5 38-f, 38-r 1.5 31-f, 31-r 2.0 6 1-f, 1-r 1.5 10A-f, 10-r 1.5 35F-f, 35F-r 1.5 34-f, 34-r 1.5 7 20-f, 20-r 1.5 7C-f, 7C-r 1.5 17F-f, 17F-r 1.5 15A-f, 15A-r 1.5 ↵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 PCRa Actual conventional serotyping result(s) (no. of positive isolates) % of total (n = 421) 1 1 (13) 3.1 3 3 (24) 5.7 4 4 (23) 5.5 6A/6B, 35 isolates 6A (19), 6B (16) 8.3 7F/(7A) 7F (20), 7A (2) 5.2 7C/(7B)/(40) 7C (8), 7B (1) 2.1 8 8 (8) 1.9 9V/(9A) 9V (15) 3.6 14 14 (20) 4.8 20 20 (6) 1.4 31 31 (8) 1.9 34 34 (7) 1.6 38/(25F) 38 (8) 1.9 10A 10A (9) 2.1 11A/(11D) 11A (10) 2.3 12F/(12A)/(44)/(46) 12F (21) 4.9 15A 15A (9) 2.1 15B/C 15B/C (15) 3.2 16F 16F (9) 2.1 17F 17F (10) 2.3 18C/(18B)/(18F)/(18A), 3 isolates 18C (8), 18B (3), 18F (1), 18A (1) 3.1 19A 19A (34) 8.1 19F 19F (10) 2.3 22F/(22A) 22F (19), 22A (1) 5.0 23F 23F (16) 3.8 33F/(33A)/(37) 33F (10) 2.4 35B 35B (8) 1.9 35F/(47F) 35F (9) 2.1 Nontypeable 9N (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).
