The FDA has recently published rules to improve on-farm microbial safety for produce typically consumed raw by the consumer, with a focus on agriculture water quality.  The rules require farmers to regularly monitor generic E. coli as an indicator of water quality, with a sample limit 235 cfu/100 ml and a rolling 5-sample mean of 126 cfu/100 ml. To meet these requirements farmers will need methods that are reliable, rapid, cost-effective, and easy-to-use.  Lateral Flow Assay (LFA) test strips have successfully been used to address these challenges in areas ranging from allergen to pregnancy testing.  Our work focuses on combining phage-based signal amplification with an LFA strip to detect generic E. coli. T7 phage is utilized due to its broad specificity for E. coli, short infection cycle, and relatively large burst size allowing for a 100-fold increase in the signal of a single E. coli cell.  The LFA strip is a sandwich based assay utilizing antibodies for the T7 phage and a fluorescent nanoparticle reporter.  A sample containing E. coli cells is inoculated 103 pfu/ml of T7 phage for 90 minutes, and then a 30�l aliquot is tested on the LFA.  Our data shows that after incubation, the final level of  T7 phage in the sample is proportional to the initial number of E. coli cells present in the sample (R2 = .96), and that a level of 103 cfu/ml of e. coli must be present to produce sufficient phage signal above our initial inoculation.  Our data also shows that the LFA can detect a level of 104 phage pfu/ml of sample in 30 minutes.  These findings suggest that a phage-based LFA device offers a solution for on-farm testing of agricultural water.  Optimization of sample concentration, such as filtration or IMS, should allow the method to meet required bacterial sensitivity.

Phage-based lateral flow assay for the detection of generic E. coli

246th National Meeting (2013)

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