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.

Multiplex logic processing isothermal diagnostic assays for an evolving virus

“…We have previously demonstrated that the ready-to-use LAMP-OSD assay mixes can be lyophilized and stored for extended durations without loss of activity. 13 These various features should minimize device intricacies, and in turn reduce cost, especially since only two protein enzymes and a single fluorophore-quencher pair are required. Most importantly, the molecular probes can be configured to capture both current and future fast-evolving targets, such as the Zika viruses, with both greater breadth and greater accuracy than existing tests.…”

“…Following 90 -120 min of amplification at 65 °C, the reactions were imaged at room temperature using an unmodified iPhone 6 and an UltraSlim-LED transilluminator (Syngene, Frederick, MD, USA). In some experiments, our previously described in-house 3D-printed imaging device 13 was used for fluorescence visualization and smartphone imaging. Briefly, this device uses Super Bright Blue 5 mm light emitting diodes (LED) (Adafruit, New York, NY, USA) to excite fluorescence.…”

“…12 One-pot LAMP-OSD assays that can work with crude samples are especially appealing for POC use. 13,14 These assays have been coupled to highly sensitive and reliable 'yes/no' output signals such as fluorescence, glucose, or hCG that can be readily read using offthe-shelf cellphones, glucometers, or pregnancy test strips, respectively. 12, 13, 15 16, 17, 18, 19 Toehold switch RNA sensors have also been used to link iNAATs with in vitro reporter protein translation, leading to colorimetric signals.…”

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

“…To overcome these barriers to the use of LAMP, we have previously adopted methods that originated in the field of nucleic acid computation: the use of strand exchange reactions that initiate at complementary single-stranded ‘toeholds’ and progresses via branch migration. The base-pairing predictability and programmability of strand exchange kinetics promotes the construction of exquisitely sequence-specific oligonucleotide strand displacement (OSD) probes for LAMP amplicons ( S2 Fig ), thereby greatly reducing the detection of non-specific amplification background [39, 41, 42]. For instance, we recorded positive coi LAMP-OSD signals from field-caught Ae.…”

“…Moreover, since our assays can accurately analyze mosquitoes several days after capture – the coi LAMP-OSD assay could for example identify mosquitoes after 3 weeks at 37 °C without desiccant – mosquitoes from remote collection outposts can potentially be analyzed even after delayed retrieval. The flexibility of assay timing is further accommodated by the fact that lyophilized LAMP-OSD reaction mixes that can be stored and deployed without cold chain [42]. These combined features make our assay platform the best tool to date for expanding vector surveillance to resource poor settings [72], especially in that the ease of use should allow minimally trained citizen scientists to participate in otherwise sophisticated public health monitoring operations in the field.…”