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

这个小型企业创新研究项目旨在开发一种名为amplidisk的创新技术，该技术将有助于检测种子中的细菌植物病原体。健康的种子对美国乃至全世界的农业都非常重要。在一个种子批次中，只要有几颗受感染的种子（0.01%）就会导致疾病流行和重大的作物损失。由于一批种子中感染种子的频率通常很低，因此需要高灵敏度的种子检测方法。目前可用的检测方法使用小比例（0.01-0.1%）的液体种子提取物，可能导致假阴性结果。通过使用一种新的富集技术，该试验将能够取样到数百倍大的种子提取物，其中目标生物的数量在Ampli-disk中增加，并通过实时PCR检测和确认。第一阶段工作的目的是证明该技术在番茄种子中检测密歇根克拉维杆菌pv的可行性。接下来的第二阶段项目将验证该程序并开发包含扩增盘和含有所有PCR成分的干珠的商业试剂盒。这个项目的商业应用将在农业领域。基于该技术的检测试剂盒的开发和销售有望消除患病种子批次和食品材料。更健康的种子可能会增加作物产量，并改善消费者的食品安全。

This Small Business Innovation Research project is to develop an innovation called Amplidisks that would assist in the detection of bacterial plant pathogens in seeds. Healthy seeds are very important to agriculture in the United States and throughout the world. Just a few infected seeds (0.01%) in a seed lot can result in a disease epidemic and significant crop loss. Since the frequency of infected seeds in a seed lot is generally very low, highly sensitive seed testing methods are needed. Currently available assays use small aliquots (0.01-0.1%) of a liquid seed extract, and can lead to false negative results. This assay would enable sampling of several hundred times larger aliquots of seed extract by using a novel enrichment technique, where the number of target organisms are increased in an Ampli-disk and detected and confirmed by real-time PCR. The objective of the Phase I work is to show the feasibility of this technique for detection of Clavibacter michiganensis pv michiganensis in tomato seeds. The follow on Phase II project will validate the procedure and develop commercial kits containing Ampli-disks and dry beads with all PCR ingredients. The commercial application of this project will be in the area of agriculture. The development and marketing of test kits based on the the proposed technique is expected to result in the elimination of diseased seed lots and food materials. Healthier seeds would likely lead to increased crop yields and to improved food safety for the consumer.