title: Studying Bordetella pertussis populations by use of SNPeX, a simple high-throughput single nucleotide polymorphism typing method.
abstract: Large outbreaks of pertussis occur despite vaccination. A first step in the analyses of outbreaks is strain typing. However, the typing of Bordetella pertussis, the causative agent of pertussis, is problematic because the available assays are insufficiently discriminatory, not unequivocal, time-consuming, and/or costly. Here, we describe a single nucleotide primer extension assay for the study of B. pertussis populations, SNPeX (single nucleotide primer extension), which addresses these problems. The assay is based on the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) at the 3' end of allele-specific poly(A)-tailed primers and subsequent analysis with a capillary DNA analyzer. Each single nucleotide polymorphism (SNP) primer has a specific length, and as a result, up to 20 SNPs can be determined in one SNPeX reaction. Importantly, PCR amplification of target DNA is not required. We selected 38 SNPeX targets from the whole-genome sequencing data of 74 B. pertussis strains collected from across the world. The SNPeX-based phylogenetic trees preserved the general tree topology of B. pertussis populations based on whole-genome sequencing, with a minor loss of details. We envisage a strategy whereby SNP types (SnpTs) are quickly identified with the SNPeX assay during an outbreak, followed by whole-genome sequencing (WGS) of a limited number of isolates representing predominant SnpTs and the incorporation of novel SNPs in the SNPeX assay. The flexibility of the SNPeX assay allows the method to evolve along with the pathogen, making it a promising method for studying outbreaks of B. pertussis and other pathogens.
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Anne
authors.initials: A
authors.lastname: Zeddeman
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Sandra
authors.initials: S
authors.lastname: Witteveen
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Marieke J
authors.initials: MJ
authors.lastname: Bart
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Marjolein
authors.initials: M
authors.lastname: van Gent
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Han G J
authors.initials: HG
authors.lastname: van der Heide
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Kees J
authors.initials: KJ
authors.lastname: Heuvelman
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
authors.forename: Leo M
authors.initials: LM
authors.lastname: Schouls
authors.affiliation: Centre for Infectious Disease Control (CIb), National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands frits.mooi@rivm.nl.
authors.forename: Frits R
authors.initials: FR
authors.lastname: Mooi
mesh_terms: D001886:Bordetella pertussis / Q000145:classification* / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D014917:Whooping Cough / Q000453:epidemiology / Q000382:microbiology*