Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR

https://doi.org/10.1016/j.mimet.2018.12.021Get rights and content
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Highlights

  • Highly clonal Salmonella Heidelberg requires discriminatory subtyping methods.

  • Current subtyping by PFGE and phage typing lacks discriminatory power.

  • A novel RNase H2-dependent PCR genotyping assay meets this need.

  • Genotyping results correlate fully with WGS data in silico.

  • Potentially useful in outbreak identification, source attribution and tracking.

Abstract

We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico, modified to be RNase H2-compatible, and then optimized by laboratory testing. Optimization often required repeated cycling through variations in primer design, assay conditions, reagent concentrations and selection of alternative SNP targets. The final rhPCR assay uses 28 independent rhPCR reactions to target 14 DNA bases that can distinguish 15 possible lineages and sub-lineages of SH. On evaluation, the assay correctly identified the 12 lineages and sub-lineages represented in a panel of 75 diverse SH strains. Non-specific amplicons were observed in 160 (15.2%) of the 1050 reactions, but due to their low intensity did not compromise assay performance. Furthermore, in silico analysis of 500 closed genomes from 103 Salmonella serovars and laboratory rhPCR testing of five prevalent Salmonella serovars including SH indicated the assay can identify Salmonella isolates as SH, since only SH isolates generated amplicons from all 14 target SNPs. The genotyping results can be fully correlated with whole genome sequencing (WGS) data in silico. This fast and economical assay, which can identify SH isolates and classify them into related or unrelated lineages and sub-lineages, has potential applications in outbreak identification, source attribution and microbial source tracking.

Keywords

rhPCR
Salmonella Heidelberg
SNP genotyping
RNase-H dependent PCR

Abbreviations

BHI
brain heart infusion
dNTP
Deoxyribonucleotide triphosphate
LB
Luria-Bertani
PCR
polymerase chain reaction
PFGE
pulsed field gel electrophoresis
rhPCR
RNase-H2 dependent PCR
SH
Salmonella Heidelberg
SNP
single nucleotide polymorphism
Tm
melting temperature
WGS
whole genome sequencing

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1

These authors contributed equally to the current work.