In-field LAMP assay for rapid detection of human faecal contamination in environmental water

Increasing human population growth worldwide continues to put pressure on waterway quality. Timely diagnosis of human faecal contamination of water remains a major challenge in protecting water quality across the globe. Currently, methods of pathogen-detection in environmental waters - including culturing and polymerase chain reaction (PCR) - are relatively time-consuming, expensive, and complicated, often requiring technical expertise in a centralised laboratory. The risks to human health and the high economic impact of human faecal pollution drive the need for rapid and reliable detection methods: a field-deployable method to detect the presence of human faecal matter has the potential to dramatically streamline on-site spill-management processes. To meet this need, we optimised an in-field loop-mediated isothermal amplification assay (LAMP) based on the detection of the human-associated Bacteroides 16s rRNA marker, HF183, to specifically identify human faecal pollution in environmental waters. To purify water samples in the field, a rapid filtration protocol and lysis buffer were combined with our Bacteroides LAMP assay (Bac-LAMP). The Bac-LAMP assay can reliably detect less than 2 CFU mu L-1 in a time to positive (TP) of under 10 minutes with no off-target reaction with animal faeces (dog, cat, sheep, cow, quail and horse) commonly found in waterways. A sensitivity and specificity of 100% were seen when compared to the approved United States Environmental Protection Agency (USEPA) TaqMan HF183 qPCR assay. For the first time, this study demonstrates a simplified sampling protocol combined with a LAMP-based assay for the field detection of human faecal contamination in waterways in and around Melbourne, Australia.