Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences

Koen M Verstappen, Loes Huijbregts, Mirlin Spaninks, Jaap A Wagenaar, Ad C Fluit, Birgitta Duim

    Research output: Contribution to journalArticleAcademicpeer-review


    Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300-500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

    Original languageEnglish
    Article numbere0183925
    JournalPLoS One
    Issue number8
    Publication statusPublished - 2017


    • Animals
    • Base Sequence
    • Cat Diseases
    • Cats
    • DNA Primers
    • Diagnosis, Differential
    • Dog Diseases
    • Dogs
    • Genome, Bacterial
    • Humans
    • Phylogeny
    • Real-Time Polymerase Chain Reaction
    • Sequence Alignment
    • Sequence Analysis, DNA
    • Sequence Homology, Nucleic Acid
    • Staphylococcal Infections
    • Staphylococcus
    • Staphylococcus aureus
    • Journal Article


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