Design and Validation of New Primers for Specific and Sensitive Real-time PCR Detection and Quantification of Seven Botulinum Encoding Genes (Serotype A-G) of Clostridium botulinum

Botulinum neurotoxins (BoNTs) comprise a highly diverse group of seven serotypes (from A-G) and over 40 subtypes worldwide. Previous primer- and probe-based nucleic acid amplification tests (NAATs) for detection of BoNT encoding genes are challenged by high levels of nucleotide polymorphism both across and within subtypes. In this study, multiple BoNT gene sequences were aligned to identify highly conserved regions for the design of new primers that enable the detection of all seven serotypes under the same conditions. Specific primer sets were designed and validated using in silico, conventional and real-time PCR with constructed plasmids carrying the target fragments and spiked food matrices. The established procedure achieved highly specific and sensitive detection of BoNT serotypes A-G with sensitivity of 10 copies/reaction and a total turnaround time of approximately 1.5 hours. The procedure also eliminated the carryover PCR product by using uracil-N-glycosylase in combination with dUTP in the assay reaction mix. This study provides an alternative NAAT with higher coverage and compliments the traditional mouse bioassays in enhancing global botulism surveillance capabilities.