title: Single-plex quantitative assays for the detection and quantification of most pneumococcal serotypes.
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.
authors: Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America.
authors: Fuminori
authors: F
authors: Sakai
authors: Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America.
authors: Sopio
authors: S
authors: Chochua
authors: Pneumococcal Research, Murdoch Childrens Research Institute, The University of Melbourne Department of Paediatrics at the Royal Children's Hospital, Parkville, Victoria, Australia; Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Victoria, Australia.
authors: Catherine
authors: C
authors: Satzke
authors: Pneumococcal Research, Murdoch Childrens Research Institute, The University of Melbourne Department of Paediatrics at the Royal Children's Hospital, Parkville, Victoria, Australia.
authors: Eileen M
authors: EM
authors: Dunne
authors: Pneumococcal Research, Murdoch Childrens Research Institute, The University of Melbourne Department of Paediatrics at the Royal Children's Hospital, Parkville, Victoria, Australia; London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors: Kim
authors: K
authors: Mulholland
authors: Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America.
authors: Keith P
authors: KP
authors: Klugman
authors: Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America.
authors: Jorge E
authors: JE
authors: Vidal
mesh_terms: D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009297:Nasal Mucosa / Q000382:microbiology; D065288:Serogroup; D012703:Serotyping / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000302:isolation & purification