title: DNA microarray-based PCR ribotyping of Clostridium difficile.
abstract: This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.
authors.affiliation: Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany schneeberg.alexander@gmail.com.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schneeberg
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ehricht
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Slickers
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Vico
authors.initials: V
authors.lastname: Baier
authors.affiliation: Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Neubauer
authors.affiliation: Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zimmermann
authors.affiliation: Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, Berlin, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Rabold
authors.affiliation: Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, Berlin, Germany.
authors.forename: Antina
authors.initials: A
authors.lastname: Lübke-Becker
authors.affiliation: Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Seyboldt
mesh_terms: D000818:Animals; D016360:Clostridioides difficile / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D003015:Clostridium Infections / Q000382:microbiology / Q000662:veterinary; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D021521:Ribotyping / Q000379:methods*; D012680:Sensitivity and Specificity