A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp. in water

Abstract

Faecal pollution of water and the resulting potential presence of human enteric pathogens is a predominant threat to public health. Microbiological water quality can be assessed by the detection of standard faecal indicator bacteria (SFIB) such as E. coli or certain Enterococcus species. In recent years, isothermal amplification methods have become a useful alternative to polymerase chain reaction (PCR), allowing molecular diagnostics with simple or no instrumentation. In this study, a novel screening method for the molecular detection of Enterococcus spp. by loop-mediated isothermal amplification (LAMP) is described. A set of six specific LAMP primers was designed to amplify a diagnostic fragment of the Enterococcus 23S rRNA gene, which is present in several enterococcal species targeted by quantitative PCR (qPCR), which is the standard technique recommended by the US Environmental Protection Agency. Sensitivity and specificity tests were performed using a set of 30 Enterococcus and non-target bacterial reference strains. It is shown that LAMP is equally sensitive and even more specific than the qPCR assay. A dilution series of Enterococcus faecalis DNA revealed that the LAMP method can reliably detect 130 DNA target copies per reaction within 45 min. Additionally, enterococci isolated from Austrian surface waterbodies, as well as a set of DNA extracts from environmental waters, were tested. Contingency analysis demonstrated a highly significant correlation between the results of the developed LAMP assay and the reference qPCR method. Furthermore, a simple staining procedure with a fluorescence dye demonstrated the identification of amplified products by eye. In conclusion, this method is an important component for the efficient screening and testing of water samples in low-resource settings lacking sophisticated laboratory equipment and highly trained personnel, requiring only a simple heating block.

Keywords: Enterococcus spp.; Faecal indicator bacteria; Loop-mediated isothermal amplification; Low-resource methods; Microbiological water quality; Molecular diagnostics.