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

Vet-LIRN网络能力建设项目 FOA编号：PAR-18-604 “Vet-LIRN能力建设MALDI-MS法检测肉毒毒素” 普渡大学印第安纳州动物疾病诊断实验室 分析化学和毒理学科 项目概要/摘要 动物肉毒中毒的诊断是基于病史、临床体征和实验室确认， 小鼠生物测定（MBA）试验或定量实时聚合酶链反应（qRT-PCR）。但 MBA和qRT-PCR在检测活性肉毒杆菌毒素（BoNT）血清型方面具有一些局限性 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，疾病控制中心和 美国疾病预防控制中心（CDC）开发了一种快速，高通量，基于MALDI质谱（MS）的方法，称为 Endopep-MS，用于检测和区分样本中的活性BoNT血清型。该方法已 显示对于检测人类临床样本中的活性BoNT具有更高的灵敏度和特异性。的 本能力建设提案的目标是通过以下方式支持加强人类和动物食品安全 加强FDA Vet-LIRN实验室网络和印第安纳州毒理学部门的能力 动物疾病诊断实验室通过以下方式调查潜在的动物食源性肉毒杆菌中毒爆发 开发和验证内毒素-MS方法以检测肉毒杆菌毒素（BoNT）血清型A，B， C和D在动物食品中。该项目的第一个具体目标是优化和验证 使用CDC的Endopep-MS分析检测肉毒杆菌血清型A、B、C和D复合物。的 具体目标#1的研究设计将包括：1）通过比较 BoNT血清型A、B、C和D的肽裂解产物的实验分子量与理论分子量 裂解产物，2）确定每种血清型的检测下限和上限，以及3）确定 每种BoNT血清型储备溶液的储存稳定性。第二个具体目标是优化和 验证动物食品如宠物罐头中BoNT血清型A、B、C和D复合物的检测 食物、干草和青贮饲料。本研究将购买市售罐装宠物食品和干草。青贮 样本将从提交给ADDL的样本中获得，这些样本来自与以下疾病无关的诊断病例： 肉毒杆菌中毒或其他传染病。具体目标#2的研究设计将包括确定 对于每种食品基质中的BoNT血清型A、B、C和D，如下：1）方法特异性，2）方法 检测限，3）方法检测精度和4）每种储存的BoNT的冻融稳定性， 动物食品开发这种方法将有利于FDA Vet-LIRN和兽医诊断医学， 它将提高检测肉毒杆菌污染食品的能力 神经毒素，并有助于整体食品安全，因为动物食品事件可能标志着潜在的问题， 人类食物系统

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.