Background: Bordetella pertussis is the causative agent of pertussis (whooping cough), an acute respiratory disease. Despite high immunization coverage in Australia, pertussis has re-emerged, with current strains belonging to SNP cluster I carrying non-vaccine alleles (ptxP3/prn2), which can be further typed into 5 epidemic lineages (EL1-EL5), reflecting microevolutionary events. Increased use of PCR-based diagnostic techniques is gradually phasing out culture in many countries including Australia, creating challenges for ongoing disease surveillance. This study aimed to establish culture-independent typing in B. pertussis for continuous epidemiological surveillance of how B. pertussis is evolving against vaccine selection pressure.
Materials/methods: A multiplex PCR protocol was designed for eight targets distinguishing SNP cluster I and EL1-EL5 based on SNP in current Australian epidemic strains. Custom amplicons were amplified with Illumina adapter-linked primers and sequenced using Miseq next generation sequencing (NGS). Ten-fold serial dilutions of DNA templates were used starting from the lowest 2 ng/µl to the highest 0.2 pg/µl (DNA copy numbers 4.63x106 to 46.3 respectively).
Results: Amplification of DNA was evident in all dilutions from agarose gel electrophoresis. Bioinformatic analysis of sequenced reads showed that all reads were successfully mapped to the reference genome and the corresponding bases in the SNP targets were correctly called. All SNP targets could be detected up to 2 pg/µl and in case of the highest dilution (0.2 pg/µl), six out of eight targets were detected. At individual target level, total reads and their distribution by percentage showed ptxP3 and ampD had the best amplification. ptxP3 also had the highest coverage across all dilutions.
Conclusion: This study tested NGS of multiplex PCR products for culture-independent typing of B. pertussis. Our results found the method to be highly specific, sensitive and accurate, allowing for correct assignment of SNP cluster and epidemic lineages.