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The proposed phase I study targets the development of a simple and economical product, C-phage+, that can be used to assess ready-to-eat food products for evidence of fecal contamination. Development of C-phage+ will take into account the lessons learned from previous efforts to validate fecal contamination indicators such as coliphages.
The proposed research strategy will consider challenges such as ambiguities in scoring viral plaques caused by sample matrix effects and will also address limitations associated with extended assay turnaround times. The study will target the following objectives: validation of a simple, rapid and economical indicator system that can be used in modestly equipped laboratories; development and validation of an efficient extraction media for the recovery of coliphages from ready-to-eat produce; development of a simplified enumerative assay to quantify male-specific coliphages in the extraction buffer/eluate; validation of a pectin-based agar substitute that does not require steam sterilization and that eliminates the need for precise temperature monitoring during assay; and validation of a method to enhance visualization of viral plaques using a chromogenic substrate (e.g. X-GAL).
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Foodborne outbreaks of infectious disease have been associated with the contamination of fresh produce by infectious microorganisms such as hepatitis A virus. However, the indicator microorganisms currently used to assess the level of contamination in fresh produce are limited to bacterial microorganisms such as the total and fecal coliform groups that may have shorter half lives than the true enteric pathogens of interest. These bacterial indicators may not correlate with the presence/absence of enteric viruses effectively in fresh produce that may be consumed raw and sometimes fail to indicate contamination events. The project is to develop a simple and quantitative kit to detect coliphages as improved indicator microorganisms that more accurately index fecal contamination events. At the conclusion of the phase I study, the conceptual framework will be field-validated in a multi-state study to establish its robustness.
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An optimized elution buffer will be developed by evaluating a combination of base compounds, surfactants, and pH conditions to identify optimal recoveries of test microorganisms seeded onto model fruits and vegetables representing 'rough' and 'smooth' organic surfaces. The SMI research team will conduct carefully controlled experiments using food products seeded with known quantities of model indicators to characterize the recovery efficiency of each extraction buffer. A pectin-based agar substitute will be formulated and tested for comparison against traditional agar-based media. The utility of viral plaque enhancement techniques will be assessed by testing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-GAL) to facilitate visualization of viral plaques within the C-phage+ pectin-based medium. During the final stage of phase I study, the research team will field-validate the optimized system by collecting a variety of fruit and vegetable samples from at least four midwestern U.S. states.