Combination of Recombinase Polymerase Amplification with SYBR Green I for naked-eye, same-day detection of Escherichia coli O157:H7 in ground meat

Abstract

Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection. • Same-day detection of E. coli O157 was successful by Recombinase Polymerase Amplification (RPA). • RPA combined with SYBR Green I demonstrated suitable for naked-eye detection of E. coli O157. • The performance of the RPA combined with SYBR Green I was comparable to that of qPCR.