title: Multiplex touchdown PCR for rapid typing of the opportunistic pathogen Propionibacterium acnes.
abstract: The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45), and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses.
authors: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, United Kingdom.
authors: Emma
authors: E
authors: Barnard
authors: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors: István
authors: I
authors: Nagy
authors: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors: Judit
authors: J
authors: Hunyadkürti
authors: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, United Kingdom.
authors: Sheila
authors: S
authors: Patrick
authors: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, United Kingdom a.mcdowell@ulster.ac.uk.
authors: Andrew
authors: A
authors: McDowell
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis / Q000382:microbiology*; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011425:Propionibacterium acnes / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
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