Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing

Abstract

PCR-ribotyping, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile. z

Clostridium difficile infection: Early history, diagnosis and molecular strain typing methods

“…The method was performed with agarose gel-based electrophoresis. Bidet et al (1999) [180] improved the reading of the banding patterns by selecting a partial sequence of the rRNA genes (16Se23S) and the intergenic spacer region with a new set of primers located closer to this intergenic spacer region. The Public Health Laboratory 2 https://www.bd.com/resource.aspx?IDX¼17953.…”

Integration oferm(B)-containing elements through large chromosome fragment exchange inClostridium difficile

“…1 When specified, 50U of DNAseI (New England Biolabs) were added to the mix of cells. Resistant colonies obtained were confirmed by PCR-ribotyping 16 and PCR detection of erm(B) using primers E5 and E6. 17 Genomic DNA extraction Genomic DNA extraction was performed using the NucleoBond Ò AXG columns and NucleoBond Ò Buffer Set III (Macherey-Nagel) according to the manufacturer's instructions.…”

Detection and Enumeration ofClostridium difficileSpores in Retail Beef and Pork

“…Isolates were typed by PCR ribotyping as has been described elsewhere (3). In situations where the ribotype was known to be a recognized international ribotype from the PHLS Anaerobic Reference Unit (Cardiff, United Kingdom) by previous typing of reference strains, the appropriate numerical designation (e.g., 027) was used.…”