**4. DNA and other nucleic acid–based methods**

Numerous nucleic acid methods have been developed for detecting clostridial DNA in biological and environmental matrices. The polymerase chain reaction (PCR) to identify the presence of *C. botulinum* DNA was originally used to detect the presence of bacterial spores in samples [16]. The method is capable of detecting the presence of as few as 100 spores per reaction mixture for serotypes A, E, and F and only 10 spores per reaction mixture for serotype B.

Multiplex PCR methods have also been developed to analyze unknowns for a battery of different targets such as different pathogens and/or associated gene products of these patho‐ gens. Multiplexed assays employ different combinations or sets of PCR primers, each one specific for a gene of interest, to amplify multiple targets in one PCR tube. One such multiplex method could possibly discriminate among BoNT serotypes A, B, E, and F. As previously described, Peck et al. [16] developed a culture enrichment method that, when coupled with multiplex PCR, could identify strains of *C. botulinum* that were non-proteolytic (such as BoNT serotypes B, E, and F). This method was robust and rapid enough for use with food samples contaminated with *C. botulinum*.

Real-time PCR (RT-PCR) or quantitative PCR (qPCR) is also useful in studies of gene expres‐ sion, specifically differential expression of genes under various environmental conditions or comparative studies of different organisms. For detection of clostridial DNA, RT-PCR methods examine expression of the NTNH (non-toxic, non-hemagglutinin) and numerous other genes in *C. botulinum* serotypes A, B, E, and F [18]. Pentaplex methods have been developed to simultaneously identify and discriminate among larger numbers of different serotypes, using a wider array of different genes [19]. This technology may prove to be efficient and costeffective.

The GeneDisc Cycler is an apparatus to perform RT-PCR applications using the GeneDisc system. The GeneDisc is a disposable plastic reaction tray that is the size of a compact disc. This method has been designed for simultaneously testing for the BoNT/A, BoNT/B, BoNT/E, and BoNT/F genes. In 2011, Fach et al. evaluated the GeneDisc Cycler equipment with neurotoxin-producing clostridia and non-BoNT-producing bacteria isolated from various clinical, food, and environmental samples [20]. Results obtained using this "macroarray" were also compared to the mouse bioassay. The toxin genes were detected in all clostridial serotypes A, B, E, and F as well as in toxigenic *Clostridium baratii* Type F and toxigenic *Clostridium butyricum* Type E. No cross-reactivity was observed with bacteria not toxigenic to humans as well as *C. botulinum* Types C, D, and G. An evaluation of the GeneDisc array was performed in four European laboratories with BoNT-producing clostridia and 10 different samples that included food matrices and clinical isolates [20]. Results demonstrated the technology to be specific and reliable in the identification of *C. botulinum* cells containing genes encoding neurotoxins A, B, E, and F. Furthermore, contaminated food and fecal samples were success‐ fully tested. This assay is highly sensitive, capable of detecting as low as 5–50 genome copies in each PCR assay. The GeneDisc Cycler can also be used for monitoring neurotoxin-producing clostridia in food samples, clinical samples, and environmental matrices. A similar study was carried out examining a focused microarray for detection of genes-encoding BoNTs [21].

Recently, Kolesnikov et al. [22] described a new method called "proteolytic PCR" in which PCR is used to assay the proteolytic activity of botulinum toxin. This technology starts with DNA–protein complexes attached to a solid phase. Proteolytic cleavage releases DNA into solution. The DNA can then serve as a template for PCR. This study described its use to detect botulinum toxin and tetanus toxin proteolytic activity [22].
