ALERT-Ag++: An on-farm E. Coli analyzer for agricultural water quality monitoring

Ag water is recognized by growers and regulators as a potential conduit for microbiological contamination of produce in pre- and post-harvesting operations, as captured in the produce safety regulations contained within the FSMA. There is a very strong need in the grower community for on-farm technologies allowing rapid, remote, accurate monitoring and reporting of E.coli levels, in a simple-to-use and affordable package. The Alert-Ag++ Phase II project targets the development of a rapid automated on-farm pathogen detection system for automatically quantitating fecal indicator bacteria (E. coli) in agricultural (Ag) water, and alerting on the presence of key pathogenic serotypes. The focus of the Phase II project is the continued development and industrialization of a product line related to Ag water quality monitoring, based on results and feedback received from beta-testing during Phase I. The successfully beta-tested concept of the field analyzer integrating a disposable cartridge for E.coli analysis will be expanded to allow automatic measurement not only of the planktonic bacterial fraction, but also of the particle-attached fraction. The project will also implement the additional option of automatically field-testing for the presence of targeted pathogenic E.coli serotypes, such as STEC O157:H7, which would provide tremendous risk-assessment benefits towards the prevention of produce-related outbreaks. Finally, the project will include expanded side-by-side laboratory testing against currently-approved methods, and the development and implementation of a strategy for obtaining regulatory approval at the local, regional, and national levels.List of objectives:1. Operational field-deployable protocol for sensitive detection of the pathogenic O157:H7 E.coli serotype in Ag waterOptimize specific primers for generic E.coli and STEC O157:H7Test detection limit using generic E.coli and STEC O157:H7 synthetic DNATest lysis protocols amenable to field implementationTest protocol on lysed generic E.coli from enriched ALERT instrument cultureTest and validate on controlled surface water samples spiked with live STEC O157:H72. Disposable measurement cartridge adaptation for independent quantification of planktonic vs. particle-attached E.coli loadsDevelop automated on-cartridge filtration system for retaining attached bacteriaDevelop firmware upgrade for parallel sampling and measurement in two cartridges (with and without filter)Develop detection and data interpretation algorithm for quantifying the attached and planktonic E.coli fractions from the two cartridgesPerform side-by-side testing in laboratory conditions, against Colilert Quantitray/20003. Functional instrumentation prototypes, validation through extensive testing, industrializationDevelop and prototype nucleic acid analysis module for performing field detection of O157:H7Develop and prototype portable E.coli analyzer with autonomous operations via touch screen and embedded E.coli detection and quantification algorithmsDevelop and prototype Ag-specific installation hardware optionsPerform extensive testing in Ag-relevant locations and environmentsDesign-for-manufacturability review, define industrialization strategyDeploy Ag-specific cloud user interface and data management and reporting system4. Design and implementation of regulatory approval strategyDraft EPA side-by-side test plan at approved laboratoryPerform side-by-side mock-up preliminary tests and initial statistical analysisInitiate site-specific ATP process and coordinate with regional EPA and Water BoardPrepare test plan for local and nationwide EPA approval, engage with EPA Office of Water5. IP protection, dissemination and commercial pre-validationIntellectual property protection: drafting of patent, protection of trademarksPublish results in scientific journals and present at the relevant conferencesPresent results and technology to relevant stakeholders and potential clientsLocate regional agents and distributors for future commercialization