Simple endpoint PCR assay using portable devices for Escherichia coli quantification in water samples

Quantitative detection of Escherichia coli in water is essential for assessing fecal contamination because of its potential adverse effects on human health. Polymerase chain reaction (PCR)-based techniques enable rapid detection, facilitating timely decision-making and public health interventions. However, conventional quantitative real-time PCR (qPCR) assays generally require expensive, non-portable equipment and are often inaccessible in resource-limited settings. Therefore, this study evaluated a simple endpoint PCR combined with SYBR Green I (SGI)-based colorimetric deoxyribonucleic acid (DNA) detection (PCR-SGI) assay for E. coli quantification. In this assay, the E. coli 16S ribosomal ribonucleic acid gene was amplified by PCR using a portable thermal cycler and conventional PCR primers. Subsequently, the amplicon concentration level was estimated from SGI-induced hue changes using tablet-based image analysis with a custom sample chamber developed for stable hue data acquisition. The preliminary assay detection limit, determined using E. coli genomic DNA, was 2.47 genome copies per reaction. The assay, combined with a modified DNA extraction protocol using a commercial kit and portable devices (vortex mixer and portable centrifuge), detected E. coli in domestic wastewater and in lake and river water spiked with domestic wastewater. The E. coli concentrations obtained using the assay correlated strongly with those obtained by conventional qPCR (Pearson's correlation coefficient [r] = 0.973) and colony-forming unit (CFU) counts (r = 0.917) in samples with positive PCR results (CFU range: 10²-10⁶ CFU 100 mL^-1). Overall, the endpoint PCR-SGI assay represents a simple method for quantifying E. coli in water samples.