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

Abstract

Background Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) cause poisoning in humans with high mortality rates found in a wide variety of foods. The gold standard detection method that utilizes live mouse bioassays (MBAs) has a low detection limit, requires experienced personnel, and takes a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAAT) with species–specific target genes. Methods and results In this study, two sets of six LAMP primers each were designed based on multiple sequence alignments of the conserved regions of bont/E and bont/F genes collected from 180 serotype E strains and 30 serotype F strains published in NCBI. In silico PCR 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 other different Clostridium species and eight food poisoning isolated bacterial species. Employing the synthetic plamids, the optimal temperatures were determined for bont/E (63°C, LOD ≤ 10¹ copies/reaction) and bont/F (65°C, LOD ≤ 10² copies/reaction) within 30 minutes. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved coverage from 15–45%. 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 of C. botulinum.