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Streptococcus Pneumoniaetype Determination by Multiplex Polymerase Chain Reaction

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Streptococcus Pneumoniaetype Determination by Multiplex Polymerase Chain Reaction ORIGINAL ARTICLE Infectious Diseases, Microbiology & Parasitology DOI: 10.3346/jkms.2011.26.8.971 • J Korean Med Sci 2011; 26: 971-978 Streptococcus pneumoniae Type Determination by Multiplex Polymerase Chain Reaction Ki Wook Yun1,2, Eun Young Cho1,2, The purpose of this study was to develop pneumococcal typing by multiplex PCR and Ki Bae Hong1, Eun Hwa Choi1,2 compare it with conventional serotyping by quellung reaction. Pneumococcal strains used and Hoan Jong Lee1,2 in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children’s Hospital from 2006 to 2010. These strains were selected as 1Department of Pediatrics, Seoul National University Children’s Hospital, Seoul; 2Department of they represented 26 different serotypes previously determined by quellung reaction. Pediatrics, Seoul National University College of Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA Medicine, Seoul, Korea extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based Received: 14 March 2011 Accepted: 25 May 2011 on the presence of wciNβ and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: Address for Correspondence: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, Eun Hwa Choi, MD 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in Department of Pediatrics, Seoul National University Children’s Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-769, Korea complete concordance with those determined by conventional quellung reaction. The Tel: +82.2-2072-3624, Fax: +82.2-766-7283 multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional E-mail: [email protected] serotyping. This study was supported by a grant (no. 0420100740) from the Seoul National University Research Fund. Key Words: Streptococcus pneumoniae; Serotyping; Polymerase Chain Reaction/ methods; Korea INTRODUCTION monitoring on serotype changes of nasopharyngeal carriage and isolates from IPD is very important in making decisions on the Streptococcus pneumoniae is the leading cause of invasive and national immunization strategy. A convenient and accurate noninvasive diseases in the pediatric population and places a method for serotype determination is needed for evaluation of high burden on the health care system worldwide, including in vaccine efficacy and changes in pneumococcal epidemiology. Korea (1). Although only a limited number among the 92 sero- These have led to renewed interest in developing accurate and types account for pneumococcal diseases, the serotype distribu- efficient systems for pneumococcal serotyping. tion can vary by patient age, geographic region, and time of sur- Currently, serotype distribution is monitored by culture of the veillance (2). Currently, the licensure of the 7-valent pneumo- organism followed by serological determination of the capsular coccal conjugate vaccine (PCV7) heralds a new era in the con- type by the standard capsular test (6). The high cost of antisera, trol of pneumococcal diseases. Since the introduction of PCV7 subjectivity in interpretation, and technical expertise require- in the United States, a decrease in the incidence of invasive pneu- ments are serious drawbacks of the system. The development mococcal disease (IPD) caused by vaccine serotypes has been of PCR-based serotyping systems has the potential to overcome observed in pediatric and nonpediatric age groups (3). Also in some of the difficulties associated with serologic testing. Recent- Korea, PCV7 brought out substantial reduction in carriage of ly, the Centers for Disease Control and Prevention (CDC), Unit- vaccine serotypes and transmission of those in the community ed States, established a multiplex PCR protocol for the identifi- (4). On the other hand, extensive antibiotic use has resulted in cation of pneumococcal serotypes that can be applied to clini- increased pressure and promotion of several antibiotic-nonsus- cal or research use (7). Furthermore, the recently recognized ceptible non-PCV7 serotypes, mainly 19A (5). Thus, the com- serotype 6D can only be detected by the molecular method (8). bined pressure by antibiotic use and some replacement in na- This molecular typing method can be used for establishment of sopharyngeal carriage by PCV7 has resulted in increased car- clonal epidemiology of pneumococci (9). riage of and disease from some strains such as serotype 19A, 6C We aimed to compare pneumococcal typing using multiplex and a few others. Like this, the possibility of replacement of the PCR with the conventional quellung capsular reaction, and to vaccine types by nonvaccine types exists. Therefore, continuous detect new serotypes, 6C and 6D, by PCR and sequencing. © 2011 The Korean Academy of Medical Sciences. pISSN 1011-8934 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. eISSN 1598-6357 Yun KW, et al. • Streptococcus pneumoniae Type Determination by PCR MATERIALS AND METHODS ly, the stored clinical isolates were recovered by plating on 5% sheep blood agar plates (tryptic soy agar base supplemented Pneumococcal isolates with 5% sheep blood), and incubating in 5% CO2 at 37°C over- Hospital-wide surveillance has continued to monitor pneumo- night. A 1.5 μL aliquot of a suspension of the isolate in Mueller- coccal diseases as a part of routine clinical care at Seoul National Hinton broth and 1.5 μL antisera were mixed under a glass cov- University Children’s Hospital (SNUCH) since 1991. Each pneu- erslip and examined for capsular swelling using a phase contrast mococcal isolate was identified using standard microbiological microscope at × 1,000 magnification. tests, including colony morphology, hemolysis pattern, and op- tochin susceptibility. These isolates were all stored at -80°C un- Typing by multiplex PCR assay til use. All pneumococcal isolates used in the PCR assays were delinked Pneumococcal strains for this study included 77 isolates from with the results of serotyping by quellung reaction, and the re- the stock obtained during the period from 2006 to 2010. These searcher who performed the PCR was also blinded to these re- strains were selected to represent 26 different serotypes that were sults. Extraction and purification of DNA from pneumococcal relatively common in Korea, as recent epidemiologic studies had colonies were performed as described in the QIAamp Kit (QIA- suggested (2, 10). GEN GmbH, Hilden, Germany). Briefly, a sweep of cultures were sampled and resuspended in 100 μL distilled water in 1.7 Serotyping by quellung reaction mL microfuge tubes. These were heated at 100°C for 5 min. Af- Serotype was determined by the quellung reaction using anti- ter 500 μL nucleic lysis solution was added to each tube, the sam- serum (Statens Serum Institute, Copenhagen, Denmark). Brief- ples were incubated at 65°C for 15 min. Next, 200 μL of protein Table 1. Oligonucleotide primers used for pneumococcal typing by multiplex PCR GenBank Nucleotide Product Primers Primer sequence (5´-3´) Gene Reference accession no. position Size (bp) 1-f CR931632 CTC TAT AGA ATG GAG TAT ATA AAC TAT GGT TA wzy 9935 280 7 1-r CCA AAG AAA ATA CTA ACA TTA TCA CAA TAT TGG C 10181 3-f CR931634 ATG GTG TGA TTT CTC CTA GAT TGG AAA GTA G galU 9020 371 7 3-r CTT CTC CAA TTG CTT ACC AAG TGC AAT AAC G 9360 4-f CR931635 CTG TTA CTT GTT CTG GAC TCT CGA TAA TTG G wzy 9596 430 7 4-r GCC CAC TCC TGT TAA AAT CCT ACC CGC ATT G 9995 5-f CR931637 ATA CCT ACA CAA CTT CTG ATT ATG CCT TTG TG wzy 6123 362 7 5-r GCT CGA TAA ACA TAA TCA ATA TTT GAA AAA GTA TG 6450 6A/6B/6C-f CR931639 AAT TTG TAT TTT ATT CAT GCC TAT ATC TGG wciP 8656 250 7 6A/6B/6C-r TTA GCG GAG ATA ATT TAA AAT GAT GAC TA 8877 7C/(7B/40)-f CR931642 CTA TCT CAG TCA TCT ATT GTT AAA GTT TAC GAC GGG A wcwL 9438 260 7 7C/(7B/40)-r GAA CAT AGA TGT TGA GAC ATC TTT TGT AAT TTC 9665 7F/7A-f CR931643 CCT ACG GGA GGA TAT AAA ATT ATT TTT GAG wzy 13356 826 7 7F/7A-r CAA ATA CAC CAC TAT AGG CTG TTG AGA CTA AC 14150 9N/9L-f CR931647 GAA CTG AAT AAG TCA GAT TTA ATC AGC wzx 11948 516 20 9N/9L-r ACC AAG ATC TGA CGG GCT AAT CAA T 12439 9V/9A-f CR931648 GGG TTC AAA G TC AGA CAG TG A ATC TTA A wzy 9966 816 11 9V/9A-r CCA TGA ATG A AA TCA ACA TT G TCA GTA GC 10753 10A-f CR931649 GGT GTA GAT TTA CCA TTA GTG TCG GCA GAC wcrG 12423 628 7 10A-r GAA TTT CTT CTT TAA GAT TCG GAT ATT TCT C 13020 11A/11D-f CR931653 GGA CAT GTT CAG GTG ATT TCC CAA TAT AGT G wzy 11640 463 7 11A/11D-r GAT TAT GAG TGT AAT TTA TTC CAA CTT CTC CC 12071 12F/(12A/44/46)-f CR931660 GCA ACA AAC GGC GTG AAA GTA GTT G wzx 14407 376 7 12F/(12A/44/46)-r CAA GAT GAA TAT CAC TAC CAA TAA CAA AAC 14753 13-f CR931661 TAC TAA GGT AAT CTC TGG AAA TCG AAA GG wzx 14005 655 11 13-r CTC ATG CAT TTT ATT AAC CG C TTT TTG TTC 14630 14-f CR931662 GAA ATG TTA CTT GGC GCA GGT GTC AGA ATT wzy 7959 189 20 14-r GCC AAT ACT TCT TAG TCT CTC AGA TGA AT 8119 15A/15F-f CR931663 ATT AGT ACA GCT GCT GGA ATA TCT CTT C wzy 7804 434 7 15A/15F-r GAT CTA GTG AAC GTA CTA TTC CAA AC 8212 15B/15C-f CR931665 TTG GAA TTT TTT AAT TAG TGG CTT ACC TA wzy 7314 496 7 15B/15C-r CAT CCG CTT ATT AAT TGA AGT AAT CTG AAC C 7779 18/(18A/18B/18C/18F)-f CR931673 CTT AAT AGC TCT CAT TAT TCT TTT TTT AAG CC wzy 12687 573 7 18/(18A/18B/18C/18F)-r TTA TCT GTA AAC CAT ATC AGC ATC TGA AAC 13230 (continued to the next page) 972 http://jkms.org DOI: 10.3346/jkms.2011.26.8.971 Yun KW, et al.
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