Food poisoning caused by consuming foods containing toxic agents such as chemicals, toxins, and pathogenic microorganisms has a great impact on human health as well as economic development. According to the World Health Organization, botulism is the second leading cause of death among the most common poisoning agents. Botulism mainly occurs due to food contaminated with Clostridium botulinum (C. botulinum), a bacterium that produces botulinum neurotoxin (BoNT) which is classified as a group A poison, acts quickly, and has the potential to become a deadly bioweapon in terrorist attacks. BoNT is classified into 7 types (serotypes), labeled A to G. In low- and middle-income countries there is very little information about C. botulinum and botulism.
In Vietnam, food poisoning due to BoNT was first reported in July 2020, causing nearly a hundred people to be hospitalized. In the 2 years from 2020 to 2022, there have been 4 more consecutive poisoning cases, demonstrating the potential risk to public health as well as the suspicion of poisoning cases that may have been misdiagnosed before. BoNT at very low concentrations is already toxic, and the diversity of C. botulinum serotypes and food matrices present challenges for BoNT detection methods. Products that are canned, fermented or undergo low-temperature incubation are very common and pose a potential risk of BoNT poisoning, but have not received adequate attention. In addition, there are no commercial detection kits available on the market that meet the needs of detecting C. botulinum bacteria as well as BoNT toxin. Current detection methods based on nucleic acid amplification do not yet overcome two main shortcomings: the inability to distinguish between free DNA molecules and the DNA of living cells, and the lack of information regarding toxicity in strains with duplex or mosaic genes.
The project has two goals: first, to establish a real-time PCR analysis process based on an mRNA template with high sensitivity and specificity to detect the presence of Clostridium botulinum serotype A-G in food; and secondly, establish a proteolytic-PCR analysis process with high sensitivity and specificity to detect botulinum type B neurotoxin in food. The novelty of the project is demonstrated in the real-time PCR technique using a reverse transcription template from messenger RNA combined with specific primer-probe sets and new enzymes to help improve accuracy, detection limits, while minimizing false positives. In addition, the proteolytic-PCR process combines the enrichment of toxins with nanobody antibodies, the cleavage of a specific substrate complex bound to the target DNA segment by the botulinum toxin itself, and PCR will amplify the target DNA segment by many times, thereby increasing the sensitivity and specificity of the analysis. The project has 11 contents, of which 5 focus on establishing a real-time PCR process for goal 1; The next 5 contents are related to the creation of toxin enrichment complexes combined with the proteolytic-PCR process of objective 2, and finally the summarization of the project.
