, an obligate anaerobic spore-forming bacterium, produces botulinum neurotoxin (BoNT), the most potent toxin known [1
]. BoNT is classified as a Category A biothreat agent by the Centers for Disease Control and Prevention (CDC) because of its lethality and ease of production, transport and dissemination [4
]. In addition, BoNT poses several threats to the public health: first, the possibility of foodborne botulism represents a major potential health hazard that requires continual monitoring by the food industry. Second, infant botulism has been the most common form of human botulism in the United States for more than 20 years and hospitalizes approximately 80-100 U.S. infants annually [6
]. Third, cases of wound botulism due to intravenous drug use continue to increase [7
Botulism toxicity results from one of seven serologically distinct neurotoxins (types A-G) that cause a severe neuroparalytic disease characterized by descending flaccid paralysis [9
]. Rarely, unique strains of C. butyricum and C. baratii
may also cause human botulism through production and release of BoNT/E and F, respectively [10
]. The toxin acts by binding to peripheral cholinergic nerve endings and inhibiting release of acetylcholine at the neuromuscular junction. A part of the toxin is a zinc-dependent protease that cleaves target substrate proteins (SNAREs), located either on the plasma membrane or the synaptic vesicle, thereby preventing their binding, fusion and release of neurotransmitter. BoNTs cleave specific amino acids on the target proteins of the SNARE complex. BoNT/A and BoNT/E act on SNAP-25, while BoNT/C targets syntaxin as well as SNAP-25. The remaining toxin types (BoNT/B, BoNT/D, BoNT/E and BoNT/F) all act on synaptobrevin, but at different cleavage sites [12
The potential severity and lethality of the disease warrants sensitive and specific detection and serotyping of toxin and its typing to enable correct administration of serotype-specific antitoxin in a timely manner. Although treatment with Human Botulism Immune Globulin (BabyBIG®
) or equine antitoxin is based on clinical findings and should be instituted as rapidly as possible [5
], definitive microbiological diagnosis may take several days or even longer. This extended time to diagnosis occurs because detection of the bacterium and its toxin relies on toxicity assessment in mice (the mouse protection bioassay) and lengthy culture assays, which, while sensitive and specific, may be time-consuming and difficult [18
]. Moreover, the availability of the mouse protection bioassay is limited due to lack of animal facilities and reagent constraints.
A readily available rapid diagnostic test would be valuable for public health and medical management of foodborne, infant, wound, or bioterrorist botulism outbreaks. Quick, accurate diagnosis would enable the limited supply of equine or human antitoxin to be directed to affected patients, thereby allowing exposed but unaffected individuals to be reassured and spared unnecessary treatment with an equine serum product. A high-throughput assay would also be beneficial to the food industry, where the use of large quantities of mice is impractical.
Several studies have described PCR-based assays that detect the various serotypes of BoNT genes [20
]. With the advent of quantitative PCR (qPCR), further studies have reported assays that detect the toxin types (A, B, E and F) generally implicated in human illness and food contamination [27
]. However, comprehensive sequence analysis shows a high level of genetic variability within the toxin types that enables differentiation of toxin types into subtypes [32
]. Thus, existing assays may not reliably detect all known subtype variants within each botulinum toxin type.
For these reasons we have developed a novel two-step PCR-based assay that can detect both BoNT and other gene sequences located within the toxin gene complex. It is known that C. botulinum
DNA is readily attracted to botulinum neurotoxins, necessitating the use of various treatments for the removal of nucleic acids during toxin purification [34
]. These DNA sequences may be found even in highly purified protein preparations of the toxin and are therefore a reliable surrogate for the presence of BoNT, enabling rapid detection without using mice. As antitoxin doses are administered based on the serotype of toxin and clinical symptoms and not on the amount of active toxin present in the sample, the assay described here will provide the critical information needed for clinicians to treat affected patients. The first step in this procedure is a universal electrophoresis-based PCR that detects the presence of the C. botulinum
nontoxin-nonhemagglutinin (NTNH) gene, a highly conserved toxin complex gene that is found in all C. botulinum
toxin types and subtypes that has been found in all BoNT-producing C. botulinum
gene sequences examined to date [32
]. Thus, samples that contain BoNT can be identified irrespective of serotype, thereby providing comprehensive but not type-specific detection. A similar independent assay to detect NTNH has recently been reported by Rafael and Andreadis [38
]. The second step of the assay uses qPCR to determine quantitatively the specific BoNT toxin type by using seven different degenerate primer/probe pairs, one for each of the seven A-G toxin serotypes. These assays successfully detected toxin genes from 22 of the 26 known toxin subtypes.