Multiplex PCR Assay for Detection and Identification ofClostridium botulinumTypes A, B, E, and F in Food and Fecal Material

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Abstract
Botulism is diagnosed by detecting botulinum neurotoxin andClostridium botulinumcells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection ofClostridium botulinumtypes A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43C. botulinumstrains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp forC. botulinumtype A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, includingC. sporogenesand the nontoxigenic nonproteolyticC. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10−2spore/g for types A, B, and F to 10−1spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to containC. botulinumtype A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics ofC. botulinum.

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