Objectives: Botulism is a rare but potentially fatal disease generally caused by the neurotoxin produced by Clostridium botulinum. Symptoms of the disease include paralysis which is due to BoNT inhibiting neuro-transmitter release. Laboratory diagnosis of botulism relies on detecting BoNT in clinical or food specimens using in vivo tests. Diagnosis also includes isolation and identification of the bacterium which again relies on in vivo tests for detection of toxin production from the bacterium growing in vitro. We previously described the development of real-time PCR assays for BoNTA, B and E gene fragments, and here presented further evaluation data.

Methods:

DNA was extracted from faeces, enrichment cultures of naturally contaminated food and clinical samples and from colonies growing on agar plates. TaqMan-based assays for BoNTA, B and E gene fragments were performed using a 7700 Sequence Detector (Applied Biosystems). The assays were performed as a duplex reaction for BoNTA and B using FAM and VIC labelled probes, respectively, and as a monoplex for BoNTE using a single FAM labelled probe. All samples were tested by using the conventional bio-assay and results were compared with real-time PCR assay results.

Results:

PCR and bio-assay were found to be consistent in all samples except those that contained ‘silent B’ neurotoxin genes in addition to BoNTA genes. The samples tested comprised: direct examination of six faecal samples, 18 enrichment cultures for six clinical and 12 foods and 34 pure culture growing in vitro.

Conclusion:

This study is the first to report the successful identification of different C. botulinum toxin types for wild type BoNTA, B and E by using Taq-Man real-time PCR assay. This assay has already provided a useful adjunct to in vivo tests for the rapid identification of bacteria containing BoNT genes in wild type C. botulinum.