Grantee Research Project Results
Final Report: Inexpensive, Rapid and Comprehensive Virulence and Marker Gene (VMG) Analyzer for Waterborne Pathogens
EPA Contract Number: EPD10016Title: Inexpensive, Rapid and Comprehensive Virulence and Marker Gene (VMG) Analyzer for Waterborne Pathogens
Investigators: Stedtfeld, Robert D.
Small Business: AquaBioChip, LLC
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2010 through August 31, 2010
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2010) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Water and Wastewater
Description:
The objective of this project was to develop a microfluidic chip that can simultaneously detect eight waterborne pathogens (Cryptosporidium, Giardia, Salmonella, Shigella, E. coli, C. jejuni, V. cholerae, Legionella) with high specificity and sensitivity in the presence of DNA from water samples. For this objective, three tasks were identified: Task 1 was to develop a disposable ($5) microfluidic chip incorporating amplification and array-based assays; Task 2 was to design LAMP primers and probe assays to genotype 8 waterborne pathogens linked to 75% of outbreak cases in the United States from 1991 and 2002; and Task 3 consisted of validating the chip and designed assays using a low cost bench-top setup (constructed with additional funding in AquaBioChip's laboratory) for real-time reaction monitoring.
Summary/Accomplishments (Outputs/Outcomes):
Research for task 1 included development of a disposable chip with microfluidics that permit sample loaded into 4 reservoirs to be equally displaced into 64 individual 1 µL reaction wells for loop-mediated isothermal amplification (LAMP) of multiple targets. Results showed that the high surface-to-volume ratio of the hydrophobic cyclo olefin polymer (COC) chip did not inhibit or influence quantitative capability of the reaction, as a high correlation between the starting copies and time to positive amplification (TTP) was observed. Originally, AquaBioChip proposed to separate the chip into two separate sections, a multiple well compartment for amplification of target sequences, and a serpentine-shaped channel for specific detection with surface-bound oligonucleotide probes. Although the chemistry for immobilizing oligonucleotides on COC was developed, the ability to differentiate genotype with the sole use of LAMP prompted AquaBioChip to focus exclusively on an amplification chip for real-time detection of multiple targets without microarray. During the course of this study, AquaBioChip developed a robust strategy for fabricating chips that was optimized further to simplify and minimize manufacturing costs including: (1) reducing chip layout from three layers with valves to two layers without valves, which reduced cost and alignment issues; (2) eliminating the need for an aligned array of plungers to close valves; and (3) lowering material costs 25 fold per chip by switching to lower cost thermoplastic instead of COC.
For tasks 2 and 3, primers sets were designed to target more than 20 separate virulence and marker genes to identify and genotype 8 waterborne pathogens. Primers for Cryptosporidium and Giardia were designed around allele divergence. Initial observations showed that a single mutation on the 5’ end of the forward and backward inner primers reduced the time to positive amplification; however, targeting more than 1 mutation on the 5’ end completely destabilized the primer, and amplification did not occur. In addition, AquaBioChip has optimized the LAMP reaction by using a high concentration of SYTO81 dye to decrease the reaction time by 2 to 5 minutes compared to other dyes. A 5 to 9 minute reduction in time to positive reaction was observed using an inexpensive CCD with adjustable exposure compared to a commercially available real-time amplification detector. Blind sample mixtures consisting of waterborne pathogens spiked into gDNA extracted from environmental waters were used for in-depth validation. In detail, no false negative calls were made when 10 or more starting genomic copies were present in the reaction well. Although primer-dimer formation caused an increase in signal for some of the primer sets, no false positive calls were made when all targets for a given organism were considered. Overall, calls for presence/absence were in high agreement with spiked organisms for blind sample mixtures.
Conclusions:
Innovation and Commercialization:
An inexpensive, easy-to-use, portable microfluidic chip and complimenting CCD-based detector for rapid molecular detection of microbial pathogen was developed. With high analytical accuracy, less than 30-minute testing time, and ease of use, this device has the potential to become a market leader. Innovations include: (1) implementing LAMP for a user-friendly and disposable microfluidic polymeric chip that can simultaneously target multiple pathogens, (2) using the CCD detector with long exposure to reduce time to positive amplification up to 9 minutes compared to a conventional real-time thermocycler, (3) using a high concentration of SYTO dye to further decrease time to positive amplification by 5 minutes, (4) design and validation of novel assays targeting 8 waterborne pathogens, and (5) the ability to genotype Cryptosporidium and Giardia with LAMP assays designed around allelic divergence.
AquaBioChip recognizes that this technology cannot be used for monitoring specific pathogens in drinking water without some method for concentrating pathogenic organism in water. AquaBioChip plans to submit a Phase II SBIR proposal that will include a suitable means for low-cost and portable sample concentration that will allow a user to dispense pre-enriched sample into the device. If the device also can be shown or modified slightly to allow differentiation between viable (living) from non-viable cells, this tool has high potential for use in monitoring for total coliforms. This is currently the largest potential market (more than $100 million per year business) because of regulatory drinking water requirements. An added advantage for monitoring total coliforms is that only 100 ml sample concentration is required. Throughput of the microfluidic chip would allow further differentiation of contamination sources by targeting genes found solely in environmental or enteric sources and detailed information on pathogen presence. Organizations outside of AquaBioChip also have been interested in discussing this technology for a possible application in non-potable waters, which may represent a more viable market for the device (Foresight report). However, the analyzer must be able to differentiate cell viability and incorporate a simple means to concentrate water samples.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 3 publications | 3 publications in selected types | All 3 journal articles |
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Ahmad F, Seyrig G, Tourlousse DM, Stedtfeld RD, Tiedje JM, Hashsham SA. A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips. Biomedical Microdevices 2011;13(5):929-937. |
EPD10016 (Final) R833010 (Final) |
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Ahmad F, Stedtfeld RD, Waseem H, Williams MR, Cupples AM, Tiedje JM, Hashsham SA. Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in < 25 min. Journal of microbiological methods.2017;132:27-33. |
EPD10016 (Final) |
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Tourlousse DM, Ahmad F, Stedtfeld RD, Seyrig G, Tiedje JM, Hashsham SA. A polymer microfluidic chip for quantitative detection of multiple water-and foodborne pathogens using real-time fluorogenic loop-mediated isothermal amplification. Biomedical Microdevices.2012;14:769-78. |
EPD10016 (Final) |
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Supplemental Keywords:
small business, SBIR, EPA, waterborne pathogens, drinking water, drinking water analysis, drinking water monitoring, waterborne illnesses, waterborne disease outbreaks, rapid detection, loop-mediated amplification, LAMP, microarray hybridization, water quality monitoring, microfluidic chipsThe 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.