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, 9 (1), e85417
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Eight New Genomes and Synthetic Controls Increase the Accessibility of Rapid melt-MAMA SNP Typing of Coxiella Burnetii

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Eight New Genomes and Synthetic Controls Increase the Accessibility of Rapid melt-MAMA SNP Typing of Coxiella Burnetii

Edvin Karlsson et al. PLoS One.

Abstract

The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping, assay accessibility and inter-laboratory comparisons.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic tree.
Branch lengths are proportional to the total number of SNPs, which are indicated above or below the branches. Genomes sequenced in this study are in bold face. The genomic groups (I–VII) and plasmid types are denoted as described , , . CanSNP markers are depicted in red (C.1–C.15) and the corresponding MST markers are depicted in grey. The genomes Cb109, Cb175 and Z3055 became available after the time of assay design resulting in four new branches (C.12 to C.15). Although not included in this work, it demonstrates that the systematics of canSNPs easily can be extended when new genomes become available. The ancestral and derived state for C.1 and C.2 could not be determined due to the lack of sequence from a near neighbor (a root). Therefore, we could only design one assay C.1 that could be on either the C.1 or C.2 branches.
Figure 2
Figure 2. Synthetic vector controls.
Two control sequences with ancestral or derived SNP alleles for ten synonymous SNP markers (C.1 to C.11) were synthesized and cloned into the standard vector pEX-A (Fig. S2). Primer binding sites are marked in red, with the SNP states for the markers shown above them.

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References

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Publication types

Grant support

This work was performed under the auspices of the Coxiella Genome Sequencing Consortium (CGSC) (http://coxiella.net) and was funded by the Swedish Ministry of Defence and the German Bundeswehr as a part of the European Biodefense Laboratory Network (EBLN).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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