Real-time PCR assays that detect genes for botulinum neurotoxin A-G subtypes

Abstract

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories.

Keywords: Clostridium botulinum; clinical diagnostics application; food testing; highly sensitive and specific assay; real-time PCR assay; serotype A-G assay; surveillance and outbreak investigation.

Figure 1

Mismatches in forward and reverse primers and probes that predict a likely reduction in RT-PCR detection sensitivity with bont/B assays. The bont/B variant gene from Argentinean BoNT/B2 strains shows mismatches in the forward primer or probe from each of the three bont/B assays.

Figure 2

Sequence alignment of the gene fragment in the 12 bont/E subtypes between the CbE 2.693 forward and reverse primers. The alignment shows exact sequence matches in subtypes bont/E1-E7, with a single mismatch in the probe for bont/E9 and mismatches in the reverse primer for bont/E8, bont/E11, and bont/E12. The primer sites are highlighted in green and the probe site is highlighted in red.