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

这个小企业创新研究（SBIR）二期项目旨在开发一种高度敏感的基于pcr的诊断试剂盒，用于检测作物种子中的病原体。种子健康检测对于确定应排除在种子销售之外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镀以分离病原体或通过PCR鉴定。这种方法的一个主要限制是只能在琼脂板上测试一个小样本（0.1 ml），其最大灵敏度仅为每ml 10个细胞。在这个项目中，一种名为Ampli-disk的新型设备已经开发出来，可以测试4 ml的样本。此外，这种扩增盘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的准备。先前的I期研究表明，利用Ampli-disk和real-time PCR技术可以成功地检测和定量来自种子提取物的病原体。在二期项目中，目标是开发用于蔬菜作物10种最重要细菌病原体诊断试剂盒的扩增磁盘和实时PCR引物和探针。这个项目的商业应用将在农业上。该技术将在种子工业和其他细菌性疾病诊断中发挥重要作用。

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.