STUDIES ON THE LARGE SUBUNIT RIBOSOMAL-RNA GENES AND INTERGENIC SPACER REGIONS OF NONPROTEOLYTIC CLOSTRIDIUM-BOTULINUM TYPE-B, TYPE-E AND TYPE-F

The large-subunit ribosomal ribonucleic acid (23S rRNA) genes of non-proteolytic (group II) strains of Clostridium botulinum toxin types B, E and F were amplified using the polymerase chain reaction (PCR), and cloned in Escherichia coli. Sequence determination showed that the 23S rRNA genes were 2910 nucleotides in length, and comparative analysis revealed approximately 99.5 % sequence similarity. The 23S rRNA gene sequence of a strain phenotypically resembling non-proteolytic C. botulinum, except in not producing botulinal neurotoxin, was also determined and displayed 99.5 % sequence similarity with those from toxigenic strains. A diagnostic sequence within the 23S rRNA characteristic for non-proteolytic C. botulinum was identified and used for the design of an oligonucleotide probe. Molecular hybridizations with PCR-amplified rDNA targets provided a precise and reliable method of identifying non-proteolytic (or Group II) C. botulinum and closely related non-toxigenic strains.