A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F.

Clostridium botulinum is the aetiological agent of botulism, a disease marked by flaccid paralysis that can progress to asphyxiation and death. This species is defined by the production of one of the botulinum neurotoxins (BoNTs), which are the most potent toxins known. Because of their potency, these toxins have the potential to be used as biological weapons, and therefore C. botulinum has been classified as a category A select agent. There are four related but antigenically distinct BoNT types that cause disease in humans, A, B, E and F. The mouse bioassay is the current gold standard by which BoNTs are confirmed. However, this method is expensive, slow and labour-intensive. Although PCR-based assays have been used extensively for the detection of BoNT-producing bacteria in food, animals and faecal samples, and recently to help diagnose disease in humans, no real-time quantitative PCR (qPCR) assay has yet been developed that can identify and differentiate all four BoNTs that cause disease in humans. This report describes the development of a qPCR single-tube assay that uniquely identifies these four BoNTs responsible for human disease. A total of 79 C. botulinum isolates with varying toxin types was evaluated in this study, as well as numerous near-neighbours and other bacterial species. The results showed that this quadruplex assay was capable of detecting any of the four toxin genes in a given sample at a sensitivity of about 130-840 fg genomic DNA and could detect the presence of up to all four BoNT genes simultaneously in a given sample. The assay was also functional in the presence of extraneous organic matter commonly found in various environmental samples.