Single-plex quantitative assays for the detection and quantification of most pneumococcal serotypes

PLoS One. 2015 Mar 23;10(3):e0121064. doi: 10.1371/journal.pone.0121064. eCollection 2015.

Abstract

Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)2 was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Humans
  • Multiplex Polymerase Chain Reaction / methods*
  • Nasal Mucosa / microbiology
  • Serogroup*
  • Serotyping / methods*
  • Streptococcus pneumoniae / genetics*
  • Streptococcus pneumoniae / isolation & purification

Grants and funding

This study was supported by a subcontract granted to JEV (# 26180) by Murdoch Childrens Research Institute which received funds from the Bill and Melinda Gates Foundation (Grant 52099). Research at MCRI was also supported by the Victorian Government's Operational Infrastructure Support Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.