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

该小型企业创新研究（SBIR）第二阶段项目旨在开发一种高灵敏度的基于PCR的诊断试剂盒，用于检测农作物种子中的病原体。 种子健康检测对于确定应从种子销售中排除的受感染批次非常重要。由于一批种子中通常只有少数种子被感染，因此需要高度敏感的检测方法。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分离病原体或通过PCR鉴定。 该方法的主要局限性在于，只能在琼脂平板上测试少量样品（0.1 ml），其最大灵敏度仅为10个细胞/ml。 在该项目中，开发了一种名为Ampli-disk的新型设备，可以测试4 ml样品。此外，这种Ampli-disk可以根据需要储存和使用，不像琼脂板每次使用都需要新鲜制备。之前的第一阶段研究表明，种子提取物中的病原体可以通过使用Ampli-disk结合实时PCR成功地检测和定量。 在第二阶段的项目中，目标是开发扩增盘和实时PCR引物和探针，用于蔬菜作物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在农业领域。该技术将对种子工业和其他细菌性疾病诊断有用。

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.