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

Abstract

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. Abbreviations: BoNT, botulinum neurotoxin; qPCR, real-time quantitative PCR. Details of qPCR results for individual bacterial isolates are available as supplementary material with the online version of this paper.

Historical and current perspectives on Clostridium botulinum diversity

“…The availability of a large number of bont gene sequences has provided information needed for the development of detection or diagnostic assays using either conventional PCR or real-time PCR techniques [1,103,21,38,110,81,64,3,41,75]. PCR methods, particularly in conjunction with clinical evidence of botulism, provide important presumptive results that can steer laboratory investigations and reduce the demand for extensive mouse bioassay testing of samples.…”

Antibodies against a synthetic peptide designed to mimic a surface area of the H chain of botulinum neurotoxin A

“…Methods have utilized for detection the toxin's proteolytic activity of [see for example Refs. [20][21][22][23][24][25][26], molecular biology techniques [27][28][29][30][31][32], various immunological methods relying mainly on anti-toxin Abs [33][34][35][36][37][38][39][40][41][42] or on Abs in combination with other tools [43][44][45]. It should be noted that toxin serotypes exhibit different degrees of structural homology and therefore their use as immunogens produces Abs with differing levels of cross-reactivity.…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…In particular, assays able to detect bont/a, /b, /e, and /f, which are known to be pathogenic to humans, in a single reaction have been developed (Lindström et al 2001;Shin et al 2007;Kirchner et al 2010;Satterfield et al 2010;Fach et al 2011). Assays covering bont/c, /d, and their mosaic forms have been developed to meet the needs of veterinary medicine Woudstra et al 2012).…”