(J-395) Towards a Concentration-Extraction-Identification Device (CEID) for Sensitive Identification of Low-prevalence Pathogen in Environmental Samples

Introduction:: Wastewater-based epidemiology (WBE) tracks the prevalence of pathogens in a community via wastewater analysis. During the COVID-19 pandemic, WBE has provided valuable information on infection rates and concentration of SARS-CoV-2 in rural and urban settings. A key challenge in WBE is the extreme dilution of the pathogens in wastewater due to rainfall, agricultural run-offs, and industrial wastewater. This results in low and even undetectable concentrations of the viral RNA in wastewater samples. Therefore, a concentration step is recommended to achieve reliable viral signals. Common concentration methods such as polyethylene glycol precipitation, ultrafiltration, and ultracentrifugation are widely used in – and limited to - centralized laboratories. Those methods also require long processing times ( >90mins), expensive equipment such as ultracentrifugation systems and vacuum pumps and are followed by a separate extraction step that adds to the time and cost per sample. Therefore, there is a need for inexpensive and simple SARS-CoV-2 concentration/concentration methods that would be adaptable for low-facility settings and field applications. The objective of this work was to develop an integrated easy-to-use and inexpensive concentration-extraction-identification device (CEID) to detect low-prevalence pathogens e.g., SARS-CoV-2 in environmental samples. This technology is most impactful in rural and low-resource settings, where fast and low-cost environmental surveillance can lead to the democratization of public health tools and offer more equitable health care. Also, with several (re)emerging pathogens including measles and polio on the horizon, such CEIDs can help detect early and low concentrations of pathogens in environmental samples and enable timely public health responses.

Materials and Methods:: Composite wastewater samples were collected from wastewater treatment plants (WWTPs) in eastern Kentucky and transported to the lab in an ice-chest. Our standard extraction method started with the addition of 300uL lysis buffer (4M guanidinium thiocyanate, 0.01M MES, and 1:1 volume of deionized water and absolute ethanol), and 20uL of SeraSil-Mag™ beads to 200uL of the wastewater sample. The samples were then mixed and incubated for 20mins at 50C and 20mins at room temperature in a 1.5mL centrifuge tube where bead-RNA binding occurred. After washing steps, the RNA was eluted in nuclease-free water at 70C for 5mins. The identification was performed using RT-qPCR (Roche) and colorimetric loop-mediated isothermal amplification (LAMP) (New England Biolabs). The CEID operates based on immiscible filtration assisted by surface tension (IFAST). To fabricate the CEID, a laser cutter was used to cut five sequential wells to contain sample mixture (2mL sample + 3mL lysis buffer + 80uL beads), silicon oil, wash buffer, silicon oil, and nuclease-free water with respective 5, 0.1, 0.2, 0.1, and 0.1mL volumes in a 1.7mm thick PMMA sheet. The wells were connected via engraved channels (500um in depth) and then sealed using a PCR adhesive tape (Figure 1). Immediately after adding the sample mixture, the beads were aggregated and moved through the connecting channels and across the wells using a magnet. Then, the CEID was placed on a 70C hotplate for 5mins for elution. The eluate was then collected for identification purposes as mentioned above.

Results, Conclusions, and Discussions:: A straightforward approach to increase the final concentration of the extracted RNA was to start with a higher sample volume (e.g., 2mL). However, our earlier data showed the viral RNA recovery efficiency decreased to < %10 when we increased the starting sample volume and ran the capture reaction in 15mL conical tubes (inner diameter of ~17mm). Our CEID benefited from a shallow (1.7mm) sample well, which increased the probability of RNA-bead binding and minimized the incubation time to < 1min. The recovery efficiency on 2mL sample volume in the CEID was >%75 and the final extracted viral RNA concentration increased by >7 times compared to our standard extraction method. Also, the CEID eliminated the need for complex and expensive equipment, minimized the liquid handling steps, and decreased the total concentration and extraction procedures time to less than 7 mins per sample (including the 5min elution time). To identify the viral RNA, we used isothermal colorimetric LAMP which required minimal equipment such as a hotplate. We achieved a limit of detection (LOD) of 45 RNA copies per reaction in 25uL colorimetric LAMP reaction protocol (10uL sample eluate + 15uL LAMP master mix) in less than 30 mins off the chip. We also observed that LAMP LOD is volume independent, meaning that the LAMP reaction volume can be scaled up to 125uL (50uL sample eluate + 75uL LAMP master mix) and the LOD was maintained at 45 RNA copies per reaction. Therefore, the next generation of CEID allows for the direct elution of the viral RNA in 125uL LAMP mix on the chip. This results in a sample-to-answer time of < 35mins with a sensitivity of ~23 viral RNA copies per mL of wastewater samples.

While WBE enables monitoring emerging pathogens in wastewater, the current methodologies take hours to days and cost tens of dollars per sample and have low sensitivity of >100 copies/mL of sample. We designed and tested an all-in-one viral RNA concentration-extraction-identification device (CEID) to meet the need for a fast (< 35mins), inexpensive (~$12/sample), and sensitive (23 copies/mL of sample) detection method for low-prevalence pathogens in environmental samples.


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