A Field-Deployable Droplet Digital PCR System for the Rapid Detection of Waterborne Bacterial PathogensEPA Grant Number: SU839880
Title: A Field-Deployable Droplet Digital PCR System for the Rapid Detection of Waterborne Bacterial Pathogens
Investigators: Li, Yiyan
Institution: Fort Lewis College
EPA Project Officer: Callan, Richard
Project Period: October 1, 2019 through September 30, 2020
Project Amount: $24,824
RFA: P3 Awards: A National Student Design Competition Focusing on People, Prosperity and the Planet (2019) RFA Text | Recipients Lists
Research Category: P3 Awards , P3 Challenge Area - Safe and Sustainable Water Resources
Conventional waterborne bacteria monitoring is carried out by traditional culturing methods which suffer from long turnaround time, inaccurate bacteria count, and unclear genotype characterizations. The overall goals of this proposed project are: 1) Develop a field-deployable Droplet Digital Polymerase Chain Reaction (ddPCR) system that can rapidly detect pathogenic Escherichia coli (E. coli) in the field. 2) Use this proposed system to test the water samples of the Animas River and Bear Creek in Colorado. 3) Educate communities (including the Native American tribes) in the four-corner area on the importance of water resource protection and the technologies for environmental protection.
Water quality is a measure of the condition of water relative to aquatic species or human use such as drinking, swimming, and rafting. Drinking water and recreational water have different standards and these standards define the chemical, physical, or biological characteristics of water. Water acts as a passive carrier to spread diseases such as cholera, typhoid fever, and bacillary dysentery. The standard of water quality has been classified by the concentration of E. coli in water samples. E. coli are a type of fecal coliform Gram-negative bacteria commonly found in the intestines of animals and humans. The presence of E. coli in water is a strong indication of recent sewage or animal waste contamination. E. coli normally colonize the gastrointestinal tract in humans but can cause illness if they enter the kidneys or blood. The major harmful groups of E. coli are Enterotoxigenic (ETEC), Enteropathogenic (EPEC), Enterohemorrhagic (EHEC) and Enteroinvasive (EIEC). Conventional microbiological analysis of water is carried out by assaying the presence of E. coli by the culturing method. The process of filtering, culturing, processing and analyzing the bacteria population is time-consuming and laborious, which makes it impossible to be used in the field. There is a strong demand for processing water samples in the field using a portable and rapid detection system.
PCR has been used extensively for bacteria genotype characterization. However, the water quality standards are 575 cfu/100 ml for boating and rafting, 235 cfu/100 ml for swimming, and 0 cfu/100 ml for drinking. This requires the detection resolution to be at least tens of cfu/100 ml. The current real-time PCR system cannot resolve bacteria concentration variations below 500,000 cfu/100 ml without culturing, so the original filtered water sample from the field must be cultured to enrich the bacteria population before PCR. The additional culturing process introduces errors and contaminants and has a long turnaround time.
In this project, we propose to use a portable ddPCR system to provide accurate results on both the concentration and the genotypes of the bacteria population. The benefit of using ddPCR is the single-digit resolution of the system and the short turnaround time that makes it possible to instantaneously report the water quality information in the field. The ddPCR system has a microfluidic droplet generation module to partition the water sample into millions of nanoliter-sized droplets. The number of bacteria encapsulated per droplet in these systems is dictated by Poisson statistics, and the low concentration of bacteria in the water sample will enable single-cell encapsulation. The water sample will be pre-mixed with PCR reagents before droplet encapsulation. The encapsulated bacteria are then heat-lysed and the target DNA from the bacteria is amplified during PCR. A power-efficient control system manages the entire functional modules and reports the bacteria concentration in a timely manner. Based on our preliminary results on these modules, we expect the sample-to-result process can be completed within 2 hours instead of 24-48 hours using the conventional culturing method.
The results of this study are: (1) A prototype of the proposed field-deployable ddPCR system will be developed. (2) The water quality data from Animas River and Bear Creek will be collected directly in the field using the equipment prototype. (3) The equipment design and the water quality data will be published in journal papers and conference proceedings.