PCR 1 - BioMed Central
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Multiplex PCR protocol for detection of 62 Streptococcus pneumoniae serotypes. Methods Multiplex-PCRs were performed using the QIAGEN Multiplex PCR kit according to manufacturer’s indications. For each of the multiplex- PCRs, primer mix contained 10l of each primer in a final volume of 500l with Tris-EDTA buffer 1X. Each PCR mix consisted in 15l of distilled water, 5l of the primer-mix, 25l of the 2x QIAGEN Multiplex PCR Master Mix and 5l of extracted DNA. PCR conditions were: activation for 15 min at 95ºC, 35 cycles of 30 sec at 95ºC, 90 sec at 58ºC, and 90 sec at 72ºC and a final extension step of 10 min at 72ºC. Amplification products were analyzed by agarose-gel electrophoresis and amplicon sizes were determined by visual comparison to a 100 bp lambda-ladder weight marker. Validation of the specificity of the multiplex-PCRs was done testing all primers against 83 S. pneumoniae reference isolates and against a set of four non-pneumococcal viridans group streptococci previously used for the validation of an antibody array developed by our group [1]. To all isolates and cultured samples, multiplex-PCRs 1 to 4 were performed. Appropriate positive (serotype 9V for multiplex PCR 1, serotype 19A for PCR 2, …) and negative (water) controls were included in each run. If one PCR detected simultaneously more than one serotype/serogroup, a second specific PCR (from 5 to 9) was then performed. For instance, if an amplicon of 354 bp was observed in multiplex-PCR 1, then multiplex-PCR 8 was subsequently performed to determine the serotype within serogroup 18. To discriminate serotypes within serogroup 6, the PCR described by Jin et al was used [2] Table. Primers used for the multiplex-PCR for Streptococcus pneumoniae serotyping. First round Serogroup/serotype Amplicon size Primer Forward Primer Reverse Reference PCR 1 Lyt A St 14 St 18F/A/B/C St 4 Sg 10 St 11F/A/B/C/D St 9A/V St 3 100 bp 268 bp 354 bp 430 bp 512 bp 568 bp 591 bp 818 bp LytA-F: acgcaatctagcagatgaagc 14 cpsH-F: gtctgtttattctatatacaaagaggctcc 18C wciY-F: gcatctgtacagtgtgctaattggattgaag 4 wzy-F: ctgttacttgttctggactctcgttaattgg Cap-10-F: ctgacaattcatcctattccc Cap-11-F: taagctgatttcgttagtttcc 9V cpsI-F: aagtcagacagtgaatcttaac 3 capB-F: ttgttttttgtctttattcgttg LytA-R: tgtttggttggttattcgtgc 14 cpsH-R: gcattgctacaatcgctatactagatatgc 18 Cgct-R: ctttaacatctgactttttctgttcccaac 4 wyz-R: gcccactcctgttaaaatcctacccgcattg Cap-10-R: gatagtcccatataatcctcc Cap-11-R: ctacaacaatgagataatctcc 9V cpsI-R: aaatacgaatcgctaactgcc 3 capB-R: tactgagaaccttctgcccaccttagttgc 3 4 4 4 5 5 1 4 PCR 2 St 1 St 7F/7A St 8 St 5 St 19F St 19A St 33F/33A/37 St 23A St 22F/22A 99 bp 177 bp 236 bp 344 bp 408 bp 478 bp 589 bp 688 bp 755 bp 1 wyz-F: ggagactactaaattgtaatactaacacagcg Cps-7AF-F: ttgcgacaattgttgggcag Cap-8-F: tttcacgcagactagaacag Cap-5-F: tcctgttgggagtagtgtc 19F cpsI-F: cacctaattttaatactgaggttaagattgc 19A cpsK-F: gttagtcctgttttagatttatttggtgatgt Cap-33AF-F: cgtatggctactttagctcc Cps-23A-F: tctagcaagtgacgaagatg Cap-22AF-F: cagatttcttttcctcactgg 1 wyz-R: caaggatgaataaagtaaacatataatctc Cps-7AF-R: cccacccttattcaaccaag Cap-8-R: aaaggttcttgcgtatgcag Cap-5-R: gccgctgaattcttagctc 19F cpsi-R: cataggctatcagaattttaataatatcttgc 19A cpsK-R: gagcagtcaataagatgagacgatagttag Cap-33AF-R: catcctttgtcgaagccaac Cps-23A-R: ctgagtacaaaagaagcgag Cap-22AF-R: caaaaggcaccatatagaagg 4 1 5 5 4 4 1 1 5 PCR 3 St 12F/44 St 6A/6B /6C/6D St 9L/9N St 23F St 15B/15C 138 bp 220 bp 326 bp 384 bp 456 bp Cap-12A-F: acatctttggacgaactgcc 6B wzy-F: gacgtaacaaagaactaggtgctgaaac Cap-9LN-F: agatatacttgctcgaactgg 23F cpsG-F: gtaacagttgctgtagagggaattggcttttc Cap-15BC-F: agcttattaggttgggacgg Cap-12A-R: ggtaagacatcattcgcacc 6B wzy-R: aagtatataaccacgctgtaaaactctgac Cap-9LN-R: ttagccttttcacccaagatg 23F cpsG-R: cacaacacctaacacacgatggctatatgattc Cap-15BC-R: gttctgattcctgctccaag 1 4 5 4 5 St 23B St 17F St 2 St 20 512 bp 587 bp 669 bp 874 bp Cps-23B-F: tgcatatggatttaatggtgg Cps-17F-F: gctgtttacaggctttgtatc Cap-2-F: acaggaaatggtgttactcc Cap-20-F: ataccggctgtattagctgg Cps-23B-R: tatcataacacttagcaacgg Cps-17F-R: gaacatcatcattccgtaacc Cap-2-R: tataggcagagagagactac Cap-20-R: gattgtgtggtacggtagtg 5 5 5 5 PCR 4 St 35A/35B/35C/42 St 35F/47F St 36 St 7B/7C/40 St 21 St 27 St 16A St 28F/28A St 25F/25A/38 St 13 St 31 St 16F 136 bp 228 bp 297 bp 354 bp 401 bp 453 bp 502 bp 564 bp 610 bp 658 bp 711 bp 778 bp Cap-35ABC42-F: ttatgtacatggagagagtgg Cap-35F47F-F: ggagggtgagtactcttaac Cap-36-F: tccggatctattcaatttccc Cap 7B7C40-F: ttttgccggatattttagcgg Cps-GT21-F: gaatgaccctgtatggaaag Cap-27-F: ttgttccttccatacttctgg Cap-16A-F: ttatgataatggtaacgcctc Cap-28AF-F: ggtcgaggttcctatatacc Cap-25AF38-F: acacaatgtcttattgcagcc Cps-13-F: gaaaatacgatatgctctagg Cap-31-F: gacaaaccttgcggttttac Cap-16F-F: cccaataattgttatgggagg Cap-35ABC42-R: tctggcagtgaataccttttg Cap-35F47F-R: agatgggaatagtgcgtacc Cap-36-R: cagtacaagaggtataacacc Cap 7B7C40-R: atgaatcacttcctctacctg Cps-GT21-R: tttgggtaaaccatcctctg Cap-27-R: agtaacacaaggaaggatagg Cap -16A-R: gtcagccaataagtcatatac Cap-28AF-R: agtatgaccccactaacacc Cap-25AF38-R: gagtgggaaaaaactaaaccc Cps-13-R: atcgctggattatacactatg Cap-31-R: aaatatcgaacacactcgcc Cap-16F-R: tagagtgacagaacaattggg 1 5 5 5 5 1 5 5 5 5 1 1 PCR 5. Differentiation of serotypes within serogroup 10 Sg 10 512 bp Cap-10-F: ctgacaattcatcctattccc St 10A/10C 334 bp Cap-10AC-F: tttatccttgtcgggaatagg St 10A/10B 230 bp Cap-10AB-F: ccgagcagagtatgaatattg St 10F 150 bp Cap-10F-F: tgaaacggatcctgacaacc Cap-10-R: gatagtcccatataatcctcc Cap-10AC-R: aacacttttttcgcgacttgg Cap-10AB-R: gaactctgctcaatttgtctg Cap-10F-R: ccttggatacagaccaatcc 5 5 5 5 PCR 6. Differentiation of serotypes within serogroup 11 St 11F/11A/11B/11C/11D 568 bp Cap-11-F: taagctgatttcgttagtttcc St 11A/11D 487 bp wyz 11-F: tttaactttcatggtaagtggg St 11B/11C 433 bp Cap-11BC-F: aaatcgcaagatagccttcc St 11F/11A/11C/11D 262 bp C11FACD-F: ccctttagcttgagggatag St 11A 118 bp Cap-11A-F: tgcgggtgtaaccttattgg Cap-11-R: ctacaacaatgagataatctcc wzy 11-R: actaatgctaacatcacaacag Cap-11BC-R: gagacgataccaattactcc C11FACD-R: gccattgatgatgcttctgg Cap-11A-R: gagccattttgtttcgcagg 5 5 5 5 5 PCR 7. Differentiation of serotypes 12F, 12F and 46 St 12A/46 450 bp Cap-12A46-F: attgacgtgaccgaaagtcc St 12A/12B/46 268 bp Cap-12AB46-F: aactatgaaggttgaatcgcc St 12A 205 bp Cap-12A-F: gaaattctcggcttgaccac St 12F/44 138 bp Cap-12F-F: acatctttggacgaactgcc Cap-12A46-R: ggcacaaattaattccgccc Cap-12AB46-R: tcacgtccattaatttgtgcc Cap-12A-R: ttaaccatcttgccccactc Cap-12F-R: ggtaagacatcattcgcacc 5 5 5 5 PCR 8. Differentiation of serotypes within serogroup 18 St 18A 469 bp Cap-18A-F: ctaatacggatgcagaattgg St 18F/18A/18B/18C 354 bp 18CwciY-F: gcatctgtacagtgtgctaattggattgaag St 18F 117 bp Cap-18F-F: tgatggttctactgacacatc Cap-18A-R: tgaaccatgccaagtttgaag 18Cgct-R: ctttaacatctgactttttctgttcccaac Cap-18F-R: tacctgtgttacgtgcaaaac 5 4 5 PCR 9. Differentiation of serotypes 33F, 33A and 37 St 33A 690 bp Cap-33A-F: ggttgaagtcagctaagctc St 33F/33A/37 589 bp Cap-33AF-F: cgtatggctactttagctcc St 33C 365 bp Cap-33C-F: atacaccgtaaagtctgaacc St 33B/33D 227 bp Cap-33BD-F: gctgaatctcctatttgggg Cap-33A-R: ttcttcgctacgatagagcc Cap-33AF-R: catcctttgtcgaagccaac Cap-33C-R: tacaacattcagaagtgtccc Cap-33BD-R: ctaagctcaatgtgacaggg 1 1 5 5 Second round 1. Marimon JM, Monasterio A, Ercibengoa M, Pascual J, Prieto I, Simón L, Perez-Trallero E. Antibody microarray typing, a novel technique for Streptococcus pneumoniae serotyping. J Microbiol Methods. 2010; 80:274-80. 2. Jin P, Kong F, Xiao M, Oftadeh S, Zhou F, Liu C, Russell F, Gilbert GL. First report of putative Streptococcus pneumoniae serotype 6D among nasopharyngeal isolates from Fijian children. J Infect Dis 2009; 200:1375-80 3. McAvin JC, Reilly PA, Roudabush RM, Barnes WJ, Salmen A, Jackson GW, Benigna KK, Astorga A, McCleskey FK, Huff WB, Niemeyer D, Lohman KL. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol. 2001; 39: 3446-51. 4. Brito DA, Ramirez M, de Lencastre H. Serotyping Streptococcus pneumoniae by multiplex PCR. J Clin Microbiol. 2003; 41: 2378-84. 5. This work
