DETECTION OF CLOSTRIDIUM-BOTULINUM TYPE-F USING THE POLYMERASE CHAIN-REACTION

The polymerase chain reaction (PCR) was used to amplify a portion of the Clostridium botulinum type F toxin gene. An 1137-bp fragment was amplified from 11 different strains of type F C. botulinum with primers derived from the published sequence of type F strain no. 202. This fragment was not amplified from the DNA of C. botulinum types A, B and E, or from other clostridial organisms examined. When used as a hybridization probe, the 1137-bp PCR-generated fragment generated from one of the type F strains (the proteolytic strain type F Langeland) hybridized to the PCR products from all other type F toxin-producing strains tested. Portions of fragments amplified from the type F Langeland strain were sequenced. The sequence of this strain was found to exhibit approximately 3% variation from the published sequence of the non-proteolytic type F strain no. 202. Primers designed to pair with the regions of maximum sequence variation between strain 202 and the Langeland strain gave amplification products only with DNA from type F strains that exhibited the same proteolytic properties as the strain from which the primer sequences were derived. These findings underscore the need to consider Variations in sequence when designing oligonucleotide probes and PCR primers in order to avoid false negative results.