Real-time, Isothermal Detection of Shiga toxin-producing E. coli Using Recombinase Polymerase Amplification
Authors:Sean Balkcom, Andrew Cruz, Stanley Park
Mentor:Shelton E. Murinda, Professor of Animal and Veterinarian Sciences, Cal Poly Pomona
The primary goal of this study was to assess the potential application of recombinase polymerase amplification (RPA) in detection of Shiga toxin (Stx) producing E. coli (STEC). To date, there are no published reports on use of RPA for STEC detection. STEC are a major family of foodborne pathogens of immense public health, zoonotic and economic significance in the US and worldwide. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Stx1 and 2, respectively, and were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1 or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). Primers that were an ideal length (~45 bases) for universal coamplification of stx1 and stx2 sequences using RPA exo protocols could not be found. The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2, separately; subunit B sequences were too short for fitting in both primers and probes. Between the two primer/probe sets that were evaluated, probe and primer set #8 was more efficient than set #2. It detected STEC in real-time (within 5-10 minutes at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2) and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ~5 to 50 CFU/ml were achieved in serially diluted cultures grown in Brain Heart Infusion broth. This study successfully demonstrated for the first time that RPA could be used for isothermal real-time detection of STEC.