Microbiology Animal Food Project Testing Track 3 Accelerated Sample Preparation for Microbial Pathogen Detection in Pet Food Samples

微生物学动物食品项目测试轨道 3 用于宠物食品样品中微生物病原体检测的加速样品制备

• 批准号: 10447630
• 负责人: Eduardo Ximenes
• 金额: $ 0.08万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447630
• 关键词: Microbiology; Animal; Food; Project; Testing

Accelerated Sample Preparation for Microbial Pathogen Detection in Large Number of Pet Food Samples Abstract We are submitting a proposal for Discipline A: Microbiology, track 3 for Animal Food Product testing with Dog treat food/Salmonella as a hazard to Commodity pairs. Here we combine different expertise from 2 laboratories, Laboratory of Renewable Resources Engineering (LORRE) and the Office of Indiana State Chemist (OICS), to assemble a proposal consisting in animal food sample collection, accelerated food sample preparation to bring microorganisms of interest to detection levels, FDA approved loop mediated isothermal amplification (LAMP) for Salmonella screening in animal food, and whole genome sequencing as a method for confirmation of pathogen isolates. We have further optimized our sample preparation approach, as a follow up to 2015 FDA challenge award, and we now can simultaneously concentrate and recovery microorganisms of interest in 4 samples, bringing their numbers from initially not detectable, due to be present at initial levels of 1CFU/g or lower, to detection levels. This accelerated processing of samples is complete in a time frame of about 4 hours. It consists of a microbial short enrichment in selective medium combined with an enzyme treatment, followed by a pre-filtration step to remove large particles and microfiltration to concentrate and recovery selected microorganisms to detection level. The enzyme treatment helps to break down possible biofilms formation and particles that may clog pores in the hollow fiber membrane during microfiltration. Enzymes selected are based on food matrices. In the case of the samples proposed here, proteases possible combined with lipases will be used. We have shown the enzymes do not affect microbial viability, when in the presence of their respective substrates. While we have focused on Salmonella as a microorganism of interest, our developed approach can also be applied to other microorganisms. The Office of Indiana State Chemist (OISC) microbiology laboratory has major expertise in sample collection for animal food testing. It has also many years of experience in microbiological method development, testing for antibiotic concentration, and bacteria enumeration and identification, including several years of pathogen screening for Salmonella, Listeria monocytogenes, Campylobacter and Escherichia coli (STEC) in animal feed. Last year, the OISC microbiology laboratory contributed for testing in a national wide Salmonella outbreak in pet treat pig ear in Indiana. The proposed number of samples to be tested here is 250 samples pairing dog treat/Salmonella. Expected outcomes are accelerated sample preparation, increased speed and sensitivity of sample testing, and initial insights of potential recovering and detection of difficult to culture microorganisms.

微生物病原菌检测的快速制样技术 宠物食品样本 摘要 我们正在为A学科提交一份建议书：动物性食品的微生物学，第三轨道 狗的检测将食品/沙门氏菌视为对商品对的危害。在这里我们结合在一起 再生资源工程实验室(LORE)两个实验室的不同专业知识 和印第安纳州化学家办公室(OICS)，起草一份包含动物性食品的提案 样本采集，加快食品样本准备，将感兴趣的微生物带到 检测水平，FDA批准沙门氏菌环介导等温扩增(LAMP) 动物性食品中的筛选和全基因组测序作为病原体确认的一种方法 分离株。我们进一步优化了我们的样品制备方法，作为2015年FDA的后续行动 挑战奖，我们现在可以同时浓缩和回收微生物 对4个样品感兴趣，使它们的数量从最初无法检测到，将出现在 初始水平为1CFU/g或更低，以检测水平。这种对样本的加速处理是 在大约4小时的时间内完成。它由一种微生物短时间内选择性富集物组成 介质与酶处理相结合，然后进行预过滤步骤，以去除 颗粒和微滤浓缩和回收选定的微生物进行检测 水平。酶处理有助于分解可能的生物膜形成和颗粒， 在微滤过程中可能会堵塞中空纤维膜中的气孔。选定的酶是基于 在食物基质上。在这里提出的样本的情况下，蛋白酶可能与 将使用脂肪酶。我们已经证明了这些酶不会影响微生物的生存能力，当 它们各自底物的存在。虽然我们关注的是沙门氏菌作为一种微生物 有趣的是，我们开发的方法也可以应用于其他微生物。的办公室 印第安纳州化学家(OISC)微生物学实验室在样本采集方面拥有重要的专业知识 用于动物性食品检测。在微生物方法方面也有多年的经验。 开发、抗生素浓度测试以及细菌计数和鉴定， 包括几年来对沙门氏菌、单核细胞增多性李斯特菌、 动物饲料中的弯曲杆菌和大肠杆菌(STEC)。去年，OISC微生物学 实验室参与了全国范围内宠物治疗猪耳沙门氏菌暴发的检测 印第安纳州。这里建议测试的样本数量是250个配对狗样本 治疗/沙门氏菌。预期结果是加快样品准备，提高速度和 样品检测的敏感性，以及对潜在恢复和检测困难的初步见解 培养微生物。