Development of a multiplex PCR procedure for the simultaneous detection of <i>Clostridium botulinum</i> serotypes A, B, E, F

The anaerobic, spore-forming, Gram-positive bacterium Clostridium botulinum is responsible for botulism caused by the production of the botulinum neurotoxins (BoNTs), of which four serotypes A, B, E, and F, are known to be lethal to humans. In 2020, nearly a hundred cases of botulinum poisoning were reported in Vietnam for the first time. Culture-based methods, including mouse bioassay routinely used for botulism diagnosis, may take weeks, resulting in the patients not receiving timely treatment. Thus, nucleic acid amplification tests (NAATs) using specific primers have become a promising alternative. In this study, we selected four primer pairs that would produce four distinct fragments of 240 bp, 205 bp, 140 bp, and 415 bp corresponding to 4 target genes that encode BoNT/A, /B, /E, and /F for convenient separation using regular agarose electrophoresis. The modified thermal cycle that combined annealing and extension steps allowed completion of the multiplex PCR reaction in one hour. The analytical specificity of the method was also evaluated based on two strains of the same genus Clostridium and six strains of different genus Clostridium. The detection limits of the method were determined to be 103 copies/μL for serotypes A, B and E with both pure plasmids and DNA extracted from spiked samples. The total time of the established procedure was 1.5 hours, including electrophoresis.