Drigo Ilenia, Tonon Elena, Pascoletti Simone, Anniballi Fabrizio, Kalb Suzanne R, Bano Luca
Special Bacteriology Laboratory, SCT2-Istituto Zooprofilattico Sperimentale delle Venezie, Vicolo Mazzini 4, 31020 Fontane di Villorba (TV), Italy.
National Reference Centre for Botulism, Microbiological Foodborne Hazard Unit., Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy.
Toxins (Basel). 2020 Dec 24;13(1):10. doi: 10.3390/toxins13010010.
Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66-100%), 96.08% (95% CI: 86.54-99.52%), and 93.33% (95% CI: 78.25-98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15-99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.
肉毒杆菌神经毒素（BoNTs）是已知毒性最强的生物物质之一，因此，拥有可靠、易用的BoNT检测工具是食品安全以及人类和动物健康的重要目标。毒素检测和鉴定的参考方法是小鼠生物测定法（MBA）。基于质谱技术开发了一种用于BoNT分型的EndoPep-MS方法。我们已在布鲁克基质辅助激光解吸电离飞行时间质谱仪（Bruker MALDI Biotyper）上验证并应用了EndoPep-MS方法，用于检测BoNT/C和D血清型。该方法通过使用实验性和天然污染样本进行了广泛验证，并将结果与MBA法获得的结果进行比较。总体而言，除了添加BoNT/C的粪便样本显示出与特定肽段无关的信号外，对于所有测试基质，C型和D型毒素的检测限均小于或等于每500微升两个小鼠半数致死剂量（mLD）。诊断敏感性、特异性和阳性预测值分别为100%（95%置信区间：87.66 - 100%）、96.08%（95%置信区间：86.54 - 99.52%）和93.33%（95%置信区间：78.25 - 98.20%），准确率为97.47%（95%置信区间：91.15 - 99.69%）。总之，所进行的测试表明，最初使用功能更强大的质谱仪开发的EndoPep-MS方法可应用于布鲁克MALDI Biotyper仪器，结果优异，包括用于检测BoNT/C、BoNT/D、BoNT/CD和BoNT/DC毒素的蛋白水解活性。
