Dual-Gene Isothermal Amplification Coupled with Lateral Flow Strip for On-Site Accurate Detection of <i>E. coli O157:H7</i> in Food Samples

Abstract

On-site field detection of <i>E. coli O157:H7</i> in food samples is of utmost importance, since it causes a series of foodborne diseases due to infections-associated ready-to-eat foods. Due to the instrument-free nature, recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) is well-suited for such goal. However, the high genomic similarity of different <i>E. coli</i> serotypes adds difficulty to accurate differentiation of <i>E. coli O157:H7</i> from others. Dual-gene analysis could significantly improve the serotype selectivity, but will further aggravate the RPA artifacts. To address such issue, here we proposed a protocol of dual-gene RPA-LFA, in which the target amplicons were selectively recognized by peptide nucleic acid (PNA) and T7 exonuclease (TeaPNA), thus eliminating false-positives in LFA readout. Adapting rfbE_O157 and fliC_H7 genes as the targets, dual-gene RPA-TeaPNA-LFA was demonstrated to be selective for <i>E. coli O157:H7</i> over other <i>E. coli</i> serotypes and common foodborne bacteria. The minimum detection concentration was 10 copies/μL for the genomic DNA (∼300 cfu/mL <i>E. coli O157:H7</i>), and 0.24 cfu/mL <i>E. coli O157:H7</i> in food samples after 5 h bacterial preculture. For lettuce samples contaminated with <i>E. coli O157:H7</i> (single-blind), the sensitivity and specificity of the proposed method were 85% and 100%, respectively. Using DNA releaser for fast genomic DNA extraction, the assay time could be reduced to ∼1 h, which is appealing for on-site food monitoring.