Large outbreaks of pertussis occur despite vaccination. A first step in the analyses of outbreaks is strain typing. Typing of Bordetella pertussis, the causative agent of pertussis, is problematic however, because 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, called SNPeX ( S: ingle N: ucleotide P: rimer EX: tension), 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 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 whole genome sequencing data of 74 worldwide-collected B. pertussis strains. SNPeX-based phylogenetic trees preserved the general tree topology of B. pertussis populations based on whole genome sequencing, with minor loss of details. We envisage a strategy whereby SNP types (SnpTs) are quickly identified with the SNPeX assay during an outbreak, followed by WGS of a limited number of isolates representing predominant SnpTs and 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 to study outbreaks of B. pertussis and other pathogens.
Journal:J Clin Microbiol. 2015