Development of a loop-mediated isothermal amplification method for the rapid detection of Clostridium botulinum serotypes E and F

Background: Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) found in a wide variety of foods cause poisoning in humans with high mortality rates. Mouse bioassays (MBAs), the gold standard method for BoNT detection, have a low detection limit; however, require experienced personnel and take a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAATs) with primers targeting species-specific genes.

Methods and results: In this study, for each serotype E and F, six LAMP primers were designed based on multiple sequence alignments of the conserved regions of the bont/E and bont/F genes collected from 180 serotype E strains and 23 serotype F strains published in NCBI. In silico PCR amplification with the outer primer pairs showed successful amplification of the target fragments. To validate the LAMP method, we constructed two synthetic plasmids containing the target sequences extended approximately 10-50 bp to both ends. The specificity of the primers was further evaluated using six different Clostridium species and eight strains belonging to other common food poisoning-related bacterial species. Employing the synthetic plasmids, the optimal temperatures and limits of detection (LODs) were determined for bont/E (63 °C, LOD ≤ 10¹ copies/reaction) and bont/F (65 °C, LOD ≤ 10² copies/reaction) within 30 min. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved the coverage from 15 to 45%.

Conclusions: For future directions, applications of the established method, especially with the degenerate primers, could be used as an alternative assay for the rapid and sensitive detection of C. botulinum.