MODIFICATION OF A PCR RIBOTYPING METHOD FOR APPLICATION AS A ROUTINE TYPING SCHEME FOR CLOSTRIDIUM-DIFFICILE

A modification of a PCR ribotyping procedure based on polymorphisms in the 16S-23S intergenic spacer region was evaluated for use as a typin g method for Clostridium difficile. This procedure depends on the vari ation that can occur in the intergenic space between the 16S and 23S r RNA genes of the ribosomal RNA gene complex. The primers used in this study were chosen by examining the sequence of the 16S gene of C. diff icile and the 23S gene of C. botulinum. The primers used were: CTG GGG TGA AGT CGT AAC AAG G (positions 1445-1466 in the 16S rRNA gene) and GCG CCC TTT GTA GCT TGA CC (positions 1-20 in the 23S rRNA gene) and t he PCR parameters were optimised for this primer pair. To evaluate the discriminatory power of the method, PCR ribotyping was performed on s trains of C. difficile serotyped by Delmee (serogroups A-X and sub-ser ogroups A2-A10). Each isolate gave multiple DNA bands in PCR ribotypin g and a series of products ranging in size from 260 to 585 bp in lengt h was obtained. All of the 19 different serogroups gave different band ing patterns and these patterns were reproducible. This modification o f PCR ribotyping offers several advantages over the original method an d appears to hold much promise as a method for typing wild isolates of C. difficile. (C) 1996 Academic Press.