Vet-LIRN Capacity Building Detection of Botulinum Neurotoxins by MALDI-MS

VET-LIRN网络能力建设项目 FOA#：PAR-18-604 “VET-LIRN能力建设--MALDI-MS检测肉毒毒素” 普渡大学印第安纳动物疾病诊断实验室 分析化学和毒物学组 项目摘要/摘要 动物肉毒杆菌中毒的诊断是基于病史、临床体征和使用 小鼠生物测定(MBA)试验或定量实时聚合酶链式反应(qRT-PCR)。然而， 在检测活动性肉毒毒素(BONT)血清型方面，mba和qRT-PCR有一定的局限性。 存在于临床标本和食品中。为了克服相关的限制和挑战 随着MBA、PCR和其他用于诊断肉毒杆菌中毒的检测方法的使用，美国疾病控制和预防中心 预防(CDC)开发了一种快速、高通量、基于MALDI质谱学(MS)的方法，称为 Endoep-MS，用于检测和区分样本中的活跃的BONT血清型。这种方法已经被 在检测人类临床标本中的活动性BoNTs方面，显示出更高的敏感性和特异性。这个 这项能力建设提案的目标是通过以下方式支持加强人和动物食品安全 加强FDA Vet-LIRN实验室网络和印第安纳州毒物科的能力 动物疾病诊断实验室通过以下途径调查潜在的动物食源性肉毒杆菌中毒暴发 A型、B型、B型肉毒毒素(BONT)检测方法的建立与验证 C和D在动物性食品中。该项目的第一个具体目标将是优化和验证 用CDC的Endoep-MS法检测A型、B型、C型和D型肉毒杆菌复合体。这个 针对特定目标#1的研究设计将包括：1)通过比较优化BONT血清型的检测 A型、B型、C型和D型BONT多肽裂解产物的实验相对分子质量与理论值 切割产物，2)确定每个血清型的检测下限和上限，以及3)确定 每种BONT血清型库存溶液的储存稳定性。第二个具体目标将是优化和 验证在宠物罐头等动物性食品中检测A、B、C和D复合型BONT 食物、干草和青贮料。本研究将购买市面上可买到的宠物罐头食品和干草。青贮料 样本将从提交给ADL的样本中获得，这些样本来自与以下情况无关的诊断病例 肉毒杆菌中毒或其他传染病。针对具体目标#2的研究设计将包括确定 下面针对每个食品基质中的BONT血清型A、B、C和D：1)方法特异性，2)方法 检测限，3)方法检测精度和4)每个储存的BoNTs的冻融稳定性， 动物性食品。开发这种方法将使FDA Vet-LIRN和兽医诊断医学在 它将提高检测食品中肉毒杆菌污染的能力和能力 神经毒素，并有助于整体食品安全，因为动物性食品事件可能预示着 人类的食物系统。

Vet-LIRN Network Capacity-Building Projects FOA #: PAR-18-604 “Vet-LIRN Capacity Building Detection of Botulinum Toxins by MALDI-MS” Indiana Animal Disease Diagnostic Laboratory, Purdue University Analytical Chemistry and Toxicology Section Project Summary/Abstract Diagnosis of botulism in animals is based on case history, clinical signs and laboratory confirmation using the mouse bioassay (MBA) test or quantitative real-time polymerase chain reaction (qRT-PCR). However, the MBA and qRT-PCR have some limitations with respect to detecting active, botulinum toxin (BoNT) serotypes present in clinical specimens and foodstuffs. In order to overcome the limitations and challenges associated with the MBA, PCR and other assays used for diagnosing botulism, the Centers for Disease Control and Prevention (CDC) developed a rapid, high-throughput, MALDI mass spectrometry-based (MS) method, termed Endopep-MS, for detecting and differentiating active, BoNT serotypes in specimens. This method has been shown to have higher sensitivity and specificity for detecting active, BoNTs in human, clinical specimens. The objective of this capacity-building proposal is to support enhanced human and animal food safety by strengthening the capacity of the FDA Vet-LIRN laboratory network and the Toxicology Section at the Indiana Animal Disease Diagnostic Laboratory to investigate potential, animal foodborne outbreaks of botulism through the development and validation of the Endopep-MS method to detect botulinum toxin (BoNT) serotypes A, B, C, and D in animal foodstuffs. The first specific aim of this project will be to optimize and validate the detection of botulinum serotypes A, B, C and D complexes using the CDC's Endopep-MS assay. The research design for specific aim #1 will include: 1) optimizing detection of the BoNT serotypes by comparing experimental molecular weights of peptide cleavage products for BoNT serotypes A, B, C and D to theoretical cleavage products, 2) determining the lower and upper limits of detection for each serotype and 3) determining the storage stability of each BoNT serotype stock solution. The second specific aim will be to optimize and validate detection of the BoNT serotypes A, B, C and D complexes in animal foodstuffs such as canned pet food, hay and silage. Commercially available canned pet food and hay will be purchased for this study. Silage samples will be obtained from samples submitted to the ADDL from diagnostic cases not associated with botulism or other infectious diseases. The research design for specific aim #2 will consist of determining the following for BoNT serotypes A, B, C and D in each foodstuff matrix: 1) the method specificity, 2) the method limits of detection, 3) the method detection precision and 4) the freeze-thaw stability of BoNTs in each stored, animal foodstuff. Developing this method would benefit FDA Vet-LIRN and veterinary diagnostic medicine in that it would increase the capacity and capabilities to detect contamination of foodstuffs with botulinum neurotoxins and contribute to the overall food safety as animal food events could signal potential issues in the human food system.