Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

Abstract

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

Point-of-Care Lateral Flow Detection of Viable Escherichia coli O157:H7 Using an Improved Propidium Monoazide-Recombinase Polymerase Amplification Method

“…The RPA-based assay has been reported for the detection of E. coli O157:H7 in food samples, using sets of primers targeting specific genes such as rfbE, stx1, stx2, eae, uidA, and fliC [ 29 , 34 , 35 , 50 , 51 , 52 ]. In this present study, two primer sets were selected from the previously tested primers targeting the rfbE and stx2 genes based on their reported sensitivity and specificity [ 34 , 35 ].…”

Advances in quantifying microbial contamination in potable water: Potential of fluorescence‐based sensor technology

“…False‐negative results can occur when investigating environmental samples where matrix components, such as humic acids and metals, inhibit the amplification stage (Girones et al, 2010). The degree to which matrix substances inhibit amplification is unknown; however, droplet digital PCR (ddPCR) has been shown to mitigate their effect through dilution (Ibekwe et al, 2020; Stults et al, 2001).…”

Application of digital PCR for public health-related water quality monitoring

“…2 and Supplementary Table ST1 summarize the published comparisons of the analytical performance of dPCR and qPCR for health-related water microbiology. Many earlier studies have reported that dPCR and qPCR measurements harmonize well, and both can be reliably used for monitoring various microorganisms in water (Rothrock et al, 2013;Cao et al, 2015;Cao et al, 2016;Verhaegen et al, 2016;Singh et al, 2017;Ibekwe et al, 2020;Crain et al, 2021). However, qPCR and dPCR methods can have considerable differences in performance characteristics, as the enumeration approaches between these methods are fundamentally different (Table 2).…”