Wang Shuo, Zhang Huajie, Xue Yanhua, Yang Yingchao, Yuan Liyong
National Institutes for Food and Drug Control, Beijing 102629, China.
Toxins (Basel). 2025 Sep 8;17(9):453. doi: 10.3390/toxins17090453.
Botulinum neurotoxins (BoNTs), produced by the anaerobic spore-forming bacterium Clostridium botulinum, are among the most potent known biological toxins. BoNTs cause lethal botulism via contaminated food, wound infections, or infant intestinal colonization, posing significant threats to public health. Although the mouse bioassay is still being considered as the gold standard for detecting BoNTs, its drawbacks, including the lengthy experimental duration, high costs, and ethical issues, highlight the urgent need to develop alternative methods to fulfill the detection requirements. In recent years, frequent botulism poisoning incidents haves put forward higher requirements for detection technology. On-site detection is expected to be rapid and immediate, while laboratory detection requires high sensitivity and serotype discrimination capabilities. This review comprehensively introduces current detection approaches, including mouse bioassay, cell-based assays, immunological methods, endopeptidase-mass spectrometry, biosensors, chromatography, and mass spectrometry techniques. Notably, cell-based assays have been used for the potency testing of commercialized botulinum toxin type A and are considered the most promising alternative to the mouse bioassay. Biosensors based on nanomaterials demonstrate advantages in real-time detection due to their rapid response and portability, while endopeptidase-mass spectrometry achieves high sensitivity and effective serotype identification by specifically recognizing toxin-cleaved substrates. Future works shall aim to completely replace MBA, developing a detection system suitable for multiple scenarios such as clinical diagnosis, food safety monitoring, and environmental monitoring. The detection methods should also have matrix compatibility and serotype discrimination capabilities.
肉毒杆菌神经毒素（BoNTs）由厌氧产芽孢细菌肉毒梭菌产生，是已知毒性最强的生物毒素之一。BoNTs通过受污染的食物、伤口感染或婴儿肠道定植导致致命的肉毒中毒，对公众健康构成重大威胁。尽管小鼠生物测定法仍被视为检测BoNTs的金标准，但其缺点，包括实验持续时间长、成本高和伦理问题，凸显了开发替代方法以满足检测要求的迫切需求。近年来，频繁发生的肉毒中毒事件对检测技术提出了更高的要求。现场检测需要快速即时，而实验室检测则需要高灵敏度和血清型鉴别能力。本文综述全面介绍了当前的检测方法，包括小鼠生物测定法、基于细胞的测定法、免疫方法、内肽酶-质谱法、生物传感器、色谱法和质谱技术。值得注意的是，基于细胞的测定法已用于商业化A型肉毒毒素的效价测试，被认为是小鼠生物测定法最有前途的替代方法。基于纳米材料的生物传感器由于其快速响应和便携性在实时检测方面具有优势，而内肽酶-质谱法则通过特异性识别毒素切割的底物实现高灵敏度和有效的血清型鉴定。未来的工作应旨在完全取代小鼠生物测定法，开发适用于临床诊断、食品安全监测和环境监测等多种场景的检测系统。检测方法还应具有基质兼容性和血清型鉴别能力。
