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Related Concept Videos

Sanger Sequencing01:57

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...

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Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction
04:25

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Published on: February 24, 2014

Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy.

Marcus H Leung1, Kevin Bryson, Kathrin Freystatter

  • 1Centre for Clinical Microbiology, University College London Medical School, Royal Free Campus, London, United Kingdom.

Journal of Clinical Microbiology
|May 4, 2012
PubMed
Summary

A new sequence-based method uses a single PCR primer pair to identify Streptococcus pneumoniae serotypes. This cost-effective approach aids in monitoring vaccine efficacy and detecting emerging strains.

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Area of Science:

  • Microbiology
  • Vaccinology
  • Bioinformatics

Background:

  • Pneumococcal conjugate vaccines require ongoing surveillance of circulating strains to evaluate efficacy and identify replacement serotypes.
  • Traditional serological methods for identifying Streptococcus pneumoniae are inefficient and error-prone.
  • Existing DNA-based methods have limited serotype coverage, necessitating multiple polymerase chain reaction (PCR) primers.

Purpose of the Study:

  • To develop a novel, cost-effective, and simple sequence-based method for identifying Streptococcus pneumoniae serotypes.
  • To design a single PCR primer pair capable of differentiating between pneumococcal serotypes by targeting conserved regions of the capsulation locus (cps).

Main Methods:

  • A computer algorithm was employed to analyze the cps of 92 vaccine serotypes, identifying primer pairs within conserved regions flanking variable regions.
  • In silico analysis predicted a primer pair spanning the cpsB gene could amplify 84 serotypes and differentiate 46.
  • The primer set's specificity was confirmed against other bacterial species, and its performance was evaluated using 138 pneumococcal strains covering 48 serotypes.

Main Results:

  • The developed primer set demonstrated specificity for Streptococcus pneumoniae, with no amplification in related species like S. mitis, S. oralis, and S. pseudopneumoniae.
  • In silico analysis suggested potential amplification of 84 serotypes and differentiation of 46 using a single primer pair targeting cpsB.
  • Experimental testing showed successful identification of 19 out of 22 vaccine serotypes (86%) to at least the serogroup level, including key 13-valent conjugate vaccine and replacement serotypes.

Conclusions:

  • A novel, sequence-based method utilizing a single PCR primer pair offers a cost-effective and simple approach for identifying Streptococcus pneumoniae serotypes.
  • This method accurately identifies most vaccine serotypes and has the potential to monitor vaccine efficacy and detect emerging or novel serotypes.
  • The approach demonstrates reproducibility and specificity, making it a valuable tool for ongoing pneumococcal surveillance.