Grantee Research Project Results
Open-Source Droplet Digital PCR (ddPCR) System for the Rapid and Accurate Detection of Bacteria from Environmental Water Samples
EPA Grant Number: SU840868Title: Open-Source Droplet Digital PCR (ddPCR) System for the Rapid and Accurate Detection of Bacteria from Environmental Water Samples
Investigators: Li, Yiyan , Chatterley, Christie
Institution: Fort Lewis College
EPA Project Officer: Cunniff, Sydney
Phase: I
Project Period: January 1, 2024 through April 25, 2025
Project Amount: $75,000
RFA: 20th Annual P3 Awards: A National Student Design Competition Focusing on People, Prosperity and the Planet Request for Applications (RFA) (2023) RFA Text | Recipients Lists
Research Category: Heavy Metal Contamination of Soil/Water , P3 Awards , P3 Challenge Area - Safe and Sustainable Water Resources , Urban Air Toxics
Description:
E. coli are currently the most reliable predictor of fecal bacterial contamination of surface waters. However, more specific bacteria detection, including below the species level, is needed to support microbial source tracking and detailed health-based monitoring. To effectively detect bacterial contamination from water, the Polymerase Chain Reaction (PCR) technique has been used as the gold standard for amplifying target deoxyribonucleic acids (DNAs). PCR requires a power-consuming thermocycler, a set of perishable chemical reagents, standard wet lab supplies, and benchtop imaging and screening equipment. Following a 10-hour culturing process, the sample preparation, thermocycling, and imaging procedures of a few PCR trials take at least 3 hours in a lab by trained personnel.
The limit of detection of PCR and Quantitative Polymerase Chain Reaction (qPCR) is around 100 bacteria per milli-liter which makes it impossible to enumerate bacteria accurately from environmental water samples. Given that the standards for E. coli in surface water in Colorado are 126 colony-forming unit (CFU)/100 mL (existing primary contact use), 205 CFU/100 mL (potential primary contact use), and 630 CFU/100 mL (not primary contact use) (based on EPA health-based recommendations), a single-digit sensitivity detection system is required. Droplet Digital Polymerase Chain Reaction (ddPCR) partitions a raw water sample and qPCR assay into millions of micro-droplets. The PCR amplification of the DNA targets is confined in each positive individual droplet which drastically improves the signal-to-noise ratio and enables single-cell detection.
Objective:
The overall goal of this project is to develop and validate an open-source ddPCR system to rapidly detect specific waterborne bacteria from environmental water samples. To achieve this goal, the following objectives are proposed: (1) develop the ddPCR assays to detect bacteria from environmental water samples; (2) develop a new sampling and filtering procedure to boost the bacteria recovery rate from environmental water samples; (3) develop an integrated and open-source ddPCR system for field workers and environmental engineers; (4) collaborate with environmental non-profit organizations, high schools, and Native American tribes in the four-corners area on community outreach regarding the importance of water resource protection and the technologies that support environmental conservation.
Approach:
Considering off-the-shelf ddPCR systems are not affordable to most research groups, we propose an open-source ddPCR system that integrates a house-made PCR thermocycler and optical detector into one portable box. The proposed ddPCR system costs under $1000 which is 100 times less than the commercial systems. The design files and instructions will be provided to users through GitHub. The team will test environmental water samples and run ddPCR trials through the system to evaluate the performance. The evaluation results will be published through journal and conference articles. The team will host multiple technical and educational workshops for local high school students and educate the community about water resource protection through collaboration with a local environmental non-profit, Mountain Studies Institute (MSI).
Expected Results:
The results of this study are: (1) an open-source ddPCR system validated with water samples from the Animas River in the Durango area (classified as existing primary contact use); (2) patented equipment design and the water quality data published in journal papers and conference proceedings; (3) education and collaboration opportunities for high school and college students and nearby tribal communities (prospective junior environmental professionals will be trained).
Supplemental Keywords:
ddPCR, optics, filtration, waterborne bacteria detectionProgress and Final Reports:
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.