DETECTION OF THE GENES ENCODING BOTULINUM NEUROTOXIN TYPE-A TO TYPE-EBY THE POLYMERASE CHAIN-REACTION

The polymerase chain reaction (PCR) was used as the basis for the deve lopment of highly sensitive and specific diagnostic tests for organism s harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each sero type (serotypes A through E) were adjusted for optimal amplification o f the target fragment. Each PCR assay was tested with organisms expres sing each of the botulinum neurotoxin types (types A through G), Clost ridium tetani, genetically related nontoxigenic organisms, and unrelat ed strains. Each assay was specific for the intended target. The PCR r eliably identified multiple strains having the same neurotoxin type. T he sensitivity of the test was determined with different concentration s of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associ ated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detect ion of botulinum neurotoxin-producing strains.