SBIR Phase II: Quantitative Detection of Bacterial Pathogens in Seeds by Use of a Novel Enrichment Technique Coupled with Automated Real-Time PCR

SBIR 第二阶段：利用新型富集技术结合自动实时 PCR 定量检测种子中的细菌病原体

• 批准号: 0450649
• 负责人: Parm Randhawa
• 金额: --
• 依托单位: California Seed and Plant Lab., Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0450649&HistoricalAwards=false
• 关键词: SBIR; Phase; II; Quantitative; Detection

This Small Business Innovation Research (SBIR) Phase II project aims to develop a highly sensitive PCR-based diagnostic kit for the detection of pathogens in crop seeds. Seed health testing is important in order to identify infected lots that should be excluded from seed sales. Because only a few seeds in a seed lot are usually infected, highly sensitive test methods are needed. The standard method consists of extracting the pathogen into a buffer followed by plating on selective media to isolate the pathogen or identification by PCR. A major limitation of this method is that only a small sample (0.1 ml) can be tested on an agar plate, which gives a maximum sensitivity of only 10 cells per ml. In this project, a novel device called Ampli-disk, has been developed, that allows testing of a 4 ml sample. Further, this Ampli-disk can be stored and used, as needed, unlike agar plates that require fresh preparation for each use. Prior Phase I research has shown that pathogens from seed extracts can be successfully detected and quantified by using Ampli-disk coupled with real-time PCR. In the Phase II project, the objective is to develop Ampli-disks and real-time PCR primers and probes into diagnostic kits for ten most important bacterial pathogens of vegetable crops. The commercial application of this project will be in agriculture. The proposed technology will be useful to the seed industry and in other bacterial disease diagnostics.

这项小型企业创新研究（SBIR）II期项目旨在开发一个高度敏感的基于PCR的诊断试剂盒，用于检测作物种子中的病原体。 种子健康测试对于确定应排除在种子销售中的受感染批次很重要。由于通常只感染种子中的几个种子，因此需要高度敏感的测试方法。标准方法包括将病原体提取到缓冲液中，然后在选择性培养基上铺板，以通过PCR分离病原体或鉴定。 该方法的一个主要局限性是，只能在琼脂板上测试一个小样品（0.1 mL），该样品的最大灵敏度仅为每毫升10个细胞。 在这个项目中，已经开发了一种称为Ampli-Disk的新型设备，可以测试4 mL样品。此外，与需要每种用途的新准备工作不同，可以根据需要存储和使用此放大盘。先前的I期研究表明，可以通过使用Ampli-Disk与实时PCR结合的Ampli-Disk成功检测和量化种子提取物的病原体。 在第二阶段项目中，目标是开发放大器和实时PCR引物，并将探针探测为十种植物作物的十种最重要的细菌病原体的诊断试剂盒。 该项目的商业应用将用于农业。拟议的技术将对种子工业和其他细菌疾病诊断有用。