Fast, sensitive, and reliable detection of waterborne pathogens by digital PCR after coagulation and foam concentration

Abstract

The quantification of pathogens is important for assessing water safety and preventing disease outbreaks. Culture-independent approaches, such as quantitative PCR (qPCR) and digital PCR (dPCR), are useful techniques for quantifying pathogens in water samples. However, since pathogens are usually present at low concentrations in water, it is necessary to concentrate microbial cells before extracting their DNA. Many existing microbial concentration methods are inefficient or take a long time to perform. In this study, we applied a coagulation and foam separation method to concentrate environmental water samples of between 1000 and 5000mL to 100μL of DNA (i.e., a 1-5×104-fold concentration). The concentration process took <1h. The DNA samples were then used to quantify various target pathogens using dPCR. One gene, the Shiga toxin gene (stx2) of Shiga toxin-producing Escherichia coli, was detected at 32 copies/100mL in a river water sample. The coagulation and foam concentration method followed by dPCR reported herein is a fast, sensitive, and reliable method to quantify pathogen genes in environmental water samples.