Development of loop-mediated isothermal amplification (LAMP) assays for the detection of diarrheagenic E. coli in wastewater.

Abstract

Diarrheagenic E. coli is responsible for a substantial portion of foodborne diseases globally. The use of standard diagnostic tools for the detection of diarrheagenic E. coli often hampers the establishment of robust surveillance when no expensive laboratory equipment, such as thermocyclers, is present. Loop-mediated isothermal amplification (LAMP) has shown potential to enable the resource-efficient detection of pathogens. In this study, LAMP assays with two detection modes, fluorescence-based molecular beacon (MB) and nucleic acid lateral flow (NALF), were developed for the detection of the diarrheagenic E. coli strains Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterohemorrhagic E. coli (EHEC). Duplex LAMP assays for the virulence genes eae and stx2 of STEC, EPEC, and EHEC were developed and validated against wastewater from an on-site containment as a model environmental medium for wastewater-based surveillance in non-sewered areas. The developed LAMP assays showed moderate specificity toward eae and stx2, enabling crude differentiation among STEC, EPEC, and EHEC. We assessed the sensitivity of the LAMP assays and estimated a Limit of Detection (LoD) of 10²-10³ gene copies per reaction and found moderate quantitative capability for the MB-based method. The development of LAMP assays for the specific detection of STEC, EPEC, and EHEC with two distinct detection modes provides various options for their surveillance in settings without access to thermocyclers.IMPORTANCEFoodborne diarrheagenic E. coli poses a public health threat, while the variability in transmissible agents hampers outbreak investigation. The lack of lab equipment, such as thermocyclers, in some laboratory settings obstructs the establishment of robust diagnostic tools. This study addresses the need for reliable diagnostic tools for thermocycler-independent application. Validation against wastewater from an on-site containment demonstrates detection of the targets in an environmental matrix that could provide representative epidemiological insights.