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Updated: Aug 24, 2026
Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction
Published on: February 24, 2014
PCR-based assays for detection of Streptococcus pneumoniae serotypes 3, 14, 19F and 23F in respiratory specimens
1Schneider Children's Hospital of the North Shore-Long Island Jewish Health System, 269-01 76th Ave, New Hyde Park, NY 11040, USA.
Abstract:
Current culture-based assays are insensitive for detection of simultaneous respiratory tract colonization by more than one pneumococcal serotype. Separate single-tube, nested PCR-based assays have been developed to detect Streptococcus pneumoniae serotypes 3, 14, 19F and 23F by amplifying unique DNA sequences in the capsular polysaccharide gene cluster of each serotype. Pairs of 27-32-base outer primers and 20-21-base inner primers and a 20-22-base probe were designed to amplify and detect a 200-221-base sequence by dot blotting using the labelled probe. Sensitivity of the assays was 0.01-10 fg using chromosomal DNA and < or = 1 viable cell using DNA extracted from exponential-phase bacteria. Each serotype-specific assay detected chromosomal DNA from all of five to ten clinical isolates of the homologous type and did not detect DNA sequences from any of 190-204 strains from 51-52 different serotypes or 28 non-pneumococcal bacterial strains. Sixteen throat swabs from children that had been cultured for S. pneumoniae were tested in PCR assays following DNA extraction. All of six that grew S. pneumoniae serotype 3, 14, 19F or 23F were positive in the PCR assay for the homologous serotype (and in a PCR assay for sequences in lytA, present in all pneumococci) and were negative in assays for other serotypes. Of eight culture-negative specimens in children not receiving antimicrobials, three were positive for both the lytA assay and an assay for one of the four serotypes, suggesting true positive results; in three others all five PCR assays were negative and, in the remaining two, the lytA assay was positive but each of the four assays for individual serotypes was negative, suggesting either false-positive results or presence of DNA sequences from an S. pneumoniae serotype other than 3, 14, 19F or 23F. These preliminary clinical data suggest that these PCR-based assays are sensitive and specific for detection of individual serotypes of pneumococci and may be used with respiratory tract specimens.
Insights
New PCR assays detect multiple Streptococcus pneumoniae serotypes in respiratory samples. These sensitive and specific molecular tests improve upon culture-based methods for identifying pneumococcal colonization.
Area of Science:
- Molecular Microbiology
- Bacterial Pathogenesis
- Diagnostic Assay Development
Background:
- Culture-based methods are insufficient for detecting multiple Streptococcus pneumoniae serotypes simultaneously.
- Pneumococcal serotypes 3, 14, 19F, and 23F are significant causes of respiratory infections.
- Accurate identification of pneumococcal serotypes is crucial for epidemiological surveillance and treatment.
Purpose of the Study:
- To develop and evaluate sensitive and specific single-tube, nested PCR-based assays for detecting Streptococcus pneumoniae serotypes 3, 14, 19F, and 23F.
- To assess the performance of these assays compared to traditional culture methods.
- To determine the utility of PCR assays for identifying pneumococcal serotypes directly from respiratory tract specimens.
Main Methods:
- Design of outer and inner primers and probes targeting unique DNA sequences in the capsular polysaccharide gene cluster of specific pneumococcal serotypes.
- Optimization of PCR conditions for amplifying and detecting target sequences.
- Validation of assay sensitivity and specificity using known DNA quantities and clinical isolates, including testing on human throat swabs.
Main Results:
- PCR assays demonstrated high sensitivity, detecting as little as 0.01 fg of chromosomal DNA or DNA from one viable bacterial cell.
- Each serotype-specific assay accurately identified homologous strains and showed no cross-reactivity with other pneumococcal serotypes or non-pneumococcal bacteria.
- Testing on clinical throat swabs showed concordance with culture results for positive samples and identified potential true positives in culture-negative samples.
Conclusions:
- The developed PCR-based assays are sensitive and specific for the detection of individual Streptococcus pneumoniae serotypes.
- These molecular assays offer a significant improvement over culture-based methods for identifying pneumococcal serotypes in respiratory specimens.
- The PCR assays show promise for clinical application in diagnosing pneumococcal infections and understanding colonization patterns.
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