Progress 08/15/16 to 10/14/17
Outputs
Target Audience:Due to public health concerns about food safety, when the Food Safety Modernization Act (FSMA) was signed into law in January of 2011, a produce safety rule was included. The rule was based on monitoring generic E. coli as an indicator of fecal contamination since this has been the safety standard used for environmental and drinking water and because the concentrations of the specific pathogens are too low to be detected using standard microbiological methods. The regulation states that there are to be no more than 235 colony forming units (CFU) generic E. coli per 100 ml for any single water sample and a rolling geometric mean (of five samples) of no more than 126 CFU/100 ml. Although producers and suppliers are committed to good practices to reduce the risk of contamination, agricultural water specimens must to reach a qualified E. coli testing lab ideally within six to seven hours. the time constraints for achieving quality test results, coupled with rural sample collection locations,make shipping costly and the logistics of arranging shipping difficult for rural farms. Furthermore, the turnaround time for culture methods that enumerate CFUs take from 24 to 48 hours after receipt of samples. It is likely that results of a water test collected at the time of harvest might not be received until product has been distributed to the public; a positive result can then require consequential recalls. During this reporting period VisuGen focused on reaching Colorado Produce growers, the Center for Produce Safety (CPS), and communication with the FDA. Fruit and vegetable production is almost a $300 million industry at the farm gate in Colorado with over 60,000 acres in production. There are 11,600 farms in Colorado. Several local food hubs are located in the front range area. Surface water is the primary source of water used for irrigation in the West and Mountain states. Colorado ranks third in the US in water use for irrigation with total withdrawals during growth season of 12,500 million gallons per day (USGS at http://water.usgs.gov/edu/qa-usagestateirr.html). Colorado is also one of the top states for cattle production with around 2.65 million head of cattle each year. Since cattle are known shedders of pathogenic E. coli O157:H7, the proximity of cattle production to surface waters used for irrigation is a risk factor for contamination and provides additional impetus for monitoring in addition to the regulatory requirements of FMSA. VisuGen attended and exhibited at the annual meeting of the Colorado Fruit and Vegetable Growers Association (CFVGA) and met and solicited input from growers on how they intend to comply with the FSMA law. At the CPS annual meeting held in Denver additional input was obtained at the national level. The FDA was present and VisuGen met with them directly to them of our technology in development. Based on the stakeholder input at the CPS meeting the FDA delayed the implementation of E. coli testing primarily due to the difficulty growers will have meeting the turnaround time and also because the method specified in the law (EPA method 1603) is not one that most labs perform. The FDA has now approved alternative testing methods. However, these methods still require rapid transport to a lab and use culture methods with overnight turnaround. These events support the need for the more rapid on-site testing under development by VisuGen. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?VisuGen Global is an allied member of the Colorado Fruit and Vegetable Growers Association (CFVGA). Through our membership we have contacted growers that must comply with FSMA and solicited their input on how best our technology might be utilized. In addition, VisuGen attended the CFVGA annual meetings in 2016 and 2017 and introduced our technical concept as an exhibitor. VisuGen also attended the annual meeting of the Center for Produce Safety (CPS) held in Denver in June of 2017 and networked with industry leaders. Our technology was also presented in a session at the 2017 UNC water microbiology conference. VisuGen has established a web site (www.visugenglobal.com) as well as a facebook and twitter to disseminate information on our technology. Finally, VisuGen has envisioned a unique strategy that enables early customer identification and crowd funding. H2Oprofile.com is a member only site that will simplify the calculations and record keeping the produce grower must maintain for two years for FDA FSMA compliance. The data entry uniquely links a geo-tagged photo that records the precise time, date and location of the water sampled with a macro-enabled spread sheet that automatically calculates the E. coli levels as required by the FDA. The system provides for results to be directly uploaded to the produce growers secure page and ensures results are linked to the original water sample. Finally, result review is documented by digital signature. For VisuGen this provides a means to identify our future customers once the onsite water test is available. The same data base would be used by the customer but testing would be completed directly by the grower instead of having to ship the sample to a lab. What do you plan to do during the next reporting period to accomplish the goals?This is our final phase I report but we will be submitting a phase II proposal to continue the development of our technology. The ARS Eastern Research Center for Molecular Characterization of Foodborne Pathogens has agreed to collaborate with us and we are in the process of establishing a CRADA.
Impacts
What was accomplished under these goals?
The purpose of the VisuGen Global E. coli test under development is to provide a rapid on-site water monitoring method to test environmental water or to assist produce growers establish a Microbial Water Quality Profile in compliance with the FSMA produce rule requirements. Our method will provide rapid on site testing and results to avoid costly recalls and reduce illness due to microbial contamination of produce. The produce rule of FSMA mandates that produce growers test their irrigation and process water with FDA compliance phased in over the next four years. The most significant technical hurdle for accomplishing high sensitivity tests is finding low copy numbers per unit volume of the sample; for example 126 copies of the bacterium E. coli in a glass of water (100 milliliters) that is the threshold for safe irrigation recreational water use. VisuGen has discovered a strategy to capture and concentrate genes onto a unique binding material. Furthermore, the captured genes can be visualized by an affordable and rapid method that can be performed simply on sites such as at a recreational lakes or beaches, or by produce growers using a cell phone to read and record the result.The proposed technical approach provides for direct in the field detection of E. coli contaminants in 100 milliliters of environmental water with results available in 30 minutes. The major impact of our phase I research is it proves the feasibility of a method that will provide for rapid and simple on site detection and monitoring of E. coli contaminants for produce grower compliance with FSMA. This will result in reduced foodborne infections and a safer food supply. The envisioned product uses a metal oxide solid phase material to enhance capture and concentration of nucleic acid at flow rates of 20 milliliters per minute as water passes through a microfluidic chip of specific geometric design. The captured nucleic acid is then enumerated by flow through hybridization of fluorescent micro-particles that are visualized using a cell-phone reader with internet data transmission. The objectives during our phase I research were to verify the foundational steps of nucleic acid binding to aluminum oxide, hybridization capture of micro-particles, and detection by fluorescence microscopy. We identified a manufacturer that specializes in ceramic injection molding and obtained the specific aluminum oxide material initially as a slurry material or as an existing nozzle for fluid flow through. Using these materials we confirmed that both RNA and DNA binds under specific lysis buffer formulations and showed equivalent capture relative to the XtraAmp tubes previously sold by the PI as a PCR prep tube. By changing the buffer formulation a blocking buffer condition was identified where only hybridized probes are captured. Microparticles were conjugated with streptavidin, biotinylated oligonucleotide probes specific to conserved universal ribosomal RNA sequences or specific to E. coli. E coli cells were lysed using several alternative bining buffers and binding of the released nucleic acid was confimed using PCR directly from the slurry. In initial experiments as outlined in our proposal we investigate heating using Calcium Oxide as a means to selectively release rRNA. Although we showed this to be an acceptable approach, we found that using our direct lysis buffers was as efficient and rRNA is not degraded so we have pursued this approach since it simplifies the assay. By accessing the University of Colorado core microscopy facility we then showed that probe conjugated fluorescent micro-particle bind by specific hybridization using our hybridization/blocking buffer following E coli nucleic acid captured on the aluminum oxide slurry material with hybridized fluorescent micro-particles visualized to be bound to the slurry. These experiments accomplish the specific aims specified in our grant proposal that confirm the feasiblilty of our envisioned approach. Based partially on our USDA feasibility VIsuGen was funding through a NSF phase I grant to produce a prototype of the envisioned device. Results of this independent funding has enabled us to produce a 3D printed prototype of both the flow through chip made of aluminum oxide and the cell phone viewer (see photos on our web site at www.visugenglobal.com). A final aspect of the proposed USDA research was to develop PCR and RT-PCR assays specific to E. coli. We have developed these assays and verified their sensitivity for detection of single copy E. coli by targeting rRNA that is present in stationary phase E. coli at 6000 copies per cell. We have also developed a protocol for membrane filtration of 100 milliliters of environmental water followed by bead beating and binding of the released nucleic acid onto the aluminum oxide slurry material. The nucleic acid captured from environmental samples is then amplified by RT-PCR and PCR. Our phase I research results show we can quantify both RNA and DNA from environmental water collected from several sites of the Platte River. Another aspect of these studies was to use the E. coli RNA to DNA ratio as a means to determine the level of viability. The RNA vs DNA concentrations are detected by a novel method using the differential binding to these nucleic acids to aluminum oxide for different lysis buffer formulations. In summary, our approach will enable rapid turnaround so results are available at the time water is used. On site testing avoids shipping cost and logistical problems of current culture-based methods that require shipping cooled samples to a lab within 6 hours. All current methods based on culture take 18-24 hours, thus no competitor can provide the rapid 30 minute onsite results of the VisuGen method. Our technology can potentially achieve equivalent sensitivity and specificity as culture or current lab-based molecular tests and overcomes the limitations of all current methods. Furthermore, the technical approach will enable the differential detection of live versus dead cells.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Gerdes, J and Nelson, K. 2017. Moving molecular and rapid water quality methods from research to widespread use: A non-amplification molecular probe approach. Presented at the UNC 2017 Water Microbiology Conference.
http://waterinstitute.unc.edu/conferences/watermicro/presentation-archive/
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