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是为了开发一种名为Amplidisks的创新，它将帮助检测种子中的细菌植物病原体。健康的种子对美国和全世界的农业都非常重要。一批种子中仅有几个受感染的种子(0.01%)就可能导致疾病流行和严重的作物损失。由于种子批次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。目前可用的分析方法使用小等量(0.01-0.1%)的液体种子提取物，可能会导致假阴性结果。通过使用一种新的浓缩技术，这种分析将能够对数百倍大的种子提取物进行采样，其中目标微生物的数量在扩增盘中增加，并通过实时荧光聚合酶链式反应进行检测和确认。第一阶段工作的目的是证明该技术用于检测番茄种子中的密歇根杆菌密歇根杆菌的可行性。后续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。该项目的商业应用将在农业领域。基于拟议技术的检测试剂盒的开发和销售预计将导致消除病种批次和食品原料。更健康的种子可能会增加作物产量，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.