Portable platform for rapid in-field identification of human fecal pollution in water

Abstract

Human fecal contamination of water is a public health risk. However, inadequate testing solutions frustrate timely, actionable monitoring. Bacterial culture-based methods are simple but typically cannot distinguish fecal host source. PCR assays can identify host sources but require expertise and infrastructure. To bridge this gap we have developed a field-ready nucleic acid diagnostic platform and rapid sample preparation methods that enable on-site identification of human fecal contamination within 80 min of sampling. Our platform relies on loop-mediated isothermal amplification (LAMP) of human-associated Bacteroides HF183 genetic markers from crude samples. Oligonucleotide strand exchange (OSD) probes reduce false positives by sequence specifically transducing LAMP amplicons into visible fluorescence that can be photographed by unmodified smartphones. Our assay can detect as few as 17 copies/ml of human-associated HF183 targets in sewage-contaminated water without cross-reaction with canine or feline feces. It performs robustly with a variety of environmental water sources and with raw sewage. We have also developed lyophilized assays and inexpensive 3D-printed devices to minimize cost and facilitate field application.

Keywords: DNA strand displacement probes; Fecal indicator bacteria; Fecal source identification; Isothermal nucleic acid amplification; Portable molecular diagnostics.

Fig. 1. Detection of HF183 target DNA using LAMP-OSD assays

Indicated copies of recombinant plasmids bearing the HF183 target sequence were amplified by HF183-specific LAMP-OSD assays. OSD fluorescence was measured in real-time (A and B) or imaged at endpoint using a smartphone (C and D).

Fig. 2. Rapid sample preparation and HF183 LAMP-OSD analysis of environmental and surrogate water samples

(A) Schematic depicting sample preparation method. (B and C) Smartphone images depicting endpoint HF183 LAMP-OSD fluorescence in environmental water samples prepared using two-step (filter-back flush) sample processing method (B) and in surrogate water samples prepared using the three-step (filter-wash-back flush) processing method (C). All samples labeled ‘+’ were spiked with recombinant E. coli pHF183 prior to sample processing while samples labeled ‘−’ did not receive exogenous E. coli pHF183 contaminants.

Fig. 3. Detection of endogenous bacterial genes using smartphone-imaged LAMP-OSD

LAMP primers and OSD probes specific for the E. coli chromosomal gene uspB were used to identify E. coli spiked in water. (A) Indicated (6 × 10⁵ to 6) colony forming units of E. coli were either added directly to uspB LAMP-OSD reactions (non-lysed E. coli) or subjected to heat lysis prior to LAMP-OSD analysis (heat-lysed E. coli). OSD fluorescence accumulation measured in real-time is depicted. (B) Smartphone-imaged uspB LAMP-OSD analysis of indicated colony forming units of non-lysed E. coli. Endpoint fluorescence was imaged after 90 min of LAMP amplification.

Fig. 4. HF183 LAMP-OSD analysis of sewage-contaminated water

Artificial creek water samples spiked with different amounts of two independently collected samples of primary raw sewage (A:Sewage #1 and C:Sewage #2) and processed by two-step filter back flush method were analyzed by HF183 LAMP-OSD assays. Smartphone images of OSD fluorescence at amplification endpoint are depicted. HF183 analytes in Sewage #1 and Sewage #2 samples were quantitated by HF183 TaqMan qPCR (B and D).

Fig. 5. HF183 LAMP-OSD analysis of non-human feces

(A) Endpoint smartphone image of HF183 LAMP-OSD analysis (left panel) and exemplary mTEC plate culture (right panel) of dog feces (A) or cat feces (C) contaminated water. Feces from a single dog (“1 dog”) and an equal part mix of feces from multiple dogs (“9 dogs”) were independently tested. Water samples spiked with 500 μl (D1), 200 μl (D2), or 20 μl (D3) of dog feces containing samples or with 200 μl (C1), 20 μl (C2), or 2 μl (C3) of cat feces containing samples were processed by the two-step filter-back flush method. Tubes marked ‘+’ indicate positive control with pHF183 templates while tubes marked ‘−’ indicate negative control with no templates. (B) Real-time LAMP analysis of dog feces contaminated water samples (D2 and D3) using dog feces-specific primers. Negative control with no templates and positive control with gBlock templates are depicted in blue and black traces, respectively. Amplicon accumulation is indicated by increase in EvaGreen fluorescence.

Fig. 6. Storage stability of lyophilized HF183 LAMP-OSD assay master mix

Lyophilized assay master mixes were stored at the indicated temperature for 100 days. Smartphone image depicts endpoint OSD fluorescence upon HF183 LAMP-OSD analysis of indicated copies of HF183 templates using the lyophilized master mixes.