Rapid, DNA extraction-free, detection and quantification of Escherichia coli in mussels by Most Probable Number colorimetric Loop-mediated isothermal amplification

Abstract

Escherichia coli is a very well-known microorganism typically selected as indicator of fecal contamination in foods and water. The reference method for the quantification of E. coli in live mollusks relies on Most Probable Number (MPN) followed by tube confirmation on a chromogenic medium, TBX, ISO 16649-3. Even though reliable, this approach needs a minimum of two days to be completed, making it not ideal for short shelve life foods such as seafood. In the current study the conventional TBX confirmation was replaced by a novel, colorimetric Loop-mediated isothermal amplification (LAMP) assay. The MPN-LAMP method allowed to reduce by half the turnaround time of the method, 24 vs 48 h, when compared to the standard MPN-TBX. In addition to this, given the robustness of this technique to conventional inhibitory compounds, it was possible to directly add the presumptive positive tube suspension of the tube to the reaction vessel, without DNA extraction, and interpret the results in 30–35 min, after the MPN step. This approach allowed to simplify the workflow, reduce hands-on work, and costs associated to the assay. By analyzing a total of 39 mussel samples spiked with concentration ranges from <0.42–76.82 MPN/ g, minor differences in the results were observed among both confirmatory approaches, not being these statistically significant.