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

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.