AN IMPROVED ASSAY FOR IDENTIFICATION of TYPE A CLOSTRIDIUM BOTULINUM USING the POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) was used to amplify a 1340-bp fragment from the toxigenic Clostridium botulinum type A chromosome. Two oligonucleotide primers were synthesized to bracket the sequence coding for most of the type A toxin light chain (1340-bp in size). the PCR amplified a 1340-bp fragment from the DNA of all eleven C. botulinum type A strains tested. In contrast, no DNA fragments were amplified from C. botulinum types B, E, and F or from the other Clostridial chromosomal DNA examined. the products amplifed from botulinal type A strains were digested to produce fragments of the size predicted from the known location of the Hind dIII sites within the toxin gene sequence. When used as a DNA probe, the 1340-bp PCR generated fragment from type A C. botulinum strain 73A hybridized to the PCR products from all 11 type A botulinal strains and to type A thromosomal DNA preparations.