Serotyping Streptococcus pneumoniae by multiplex PCR

Abstract

The capsule is a major virulence factor of pneumococci, and it was shown that some capsular variants are associated with antimicrobial resistance and certain types of disease. Moreover, pneumococcal capsular typing has received renewed interest since the availability of conjugate vaccines, which include serotypes frequently associated with pediatric disease. Our aim was to develop a simple, reliable, and economical method for detecting epidemiologically important serotypes present in the proposed 11-valent conjugate vaccine. We designed primers based on the sequences available for the capsular types 1, 3, 4, 6B, 14, 18C, 19F, 19A, and 23F and combined them into seven multiplex PCRs. The method involves streamlined DNA template preparation and agarose gel electrophoresis to analyze the amplification products. A total of 446 pneumococci selected from among isolates colonizing the nasopharynx of children attending day care centers in Lisbon, Portugal, were typed both by conventional immunological techniques and by multiplex PCR. Capsular types identified by the PCR method invariably produced results concordant with the conventional serotyping technique. Even when the method presented does not fully type an isolate, the PCR data can guide the experimenter when using immunological serotyping. Multiplex PCR for the analysis of pneumococci provides an accurate, expeditious, and cost-effective way of reducing the number of strains that have to be serotyped by conventional immunological techniques.

FIG. 1.

Multiplex reaction scheme. GR, group reaction. G.1 through G.6, patterns obtained with the group reaction. SR1 through SR6, specific reactions 1 through 6. A through M, name of the PCR products as defined in Table 1. Serotypes in bold are those included in the proposed 11-valent vaccine.

FIG. 2.

Group reaction. Lanes represent the different patterns generated by the group reaction as indicated. Lane M, 100-bp ladder molecular size marker. The arrows indicate the names of the PCR products as defined in Table 1.

FIG. 3.

Specific reactions. (A) Specific reaction 1. Lane 1, serotype 3 (ATCC 6303); lane 2, serotype 5 (AR314). (B) Specific reaction 2. Lane 3, serotype 19A (SSISP 19A). (C) Specific reaction 3. Lane 4, serotype 4 (TIGR4); lane 5, serotype 14 (ATCC 6314). (D) Specific reaction 4. Lane 6, serotype 6B (ATCC 6326); lane 7, serotype 23F (ATCC 6323); lane 8, serotype 19A (SSISP 19A); lane 9, 18F (SSISP 18F/1). (E) Specific reaction 5. Lane 10, serotype 1 (SSISP 1/4); lane 11, serotype 18C (SSISP 18C/1); lane 12, serotype 19F (OP5248). (F) Specific reaction 6. Lane 13, serotype 18A (SSISP 18A/2). Lanes M, 100-bp ladder molecular size markers. The arrow indicates the internal control product.

FIG. 4.

Sensitivity of multiplex PCR. Numbers above the lanes indicate numbers of CFU per 10-μl reaction mixture. Lane M, 100-bp ladder molecular size marker. The arrow indicates the internal control product.