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SYNOPSIS OF RESEARCH PROPOSAL Title: DEVELOPMENT OF AN IN-SITU CAPABLE METHOD FOR DETECTING PATHOGENIC BACTERIA IN THE ALABAMA WATER SUPPLIES Name, University and City of Principal Investigators: Ahjeong Son, Auburn University, Auburn, Alabama (P.I.) Identification and Statement of the major regional water problem and research needs: Pathogenic contamination of Alabama surface water As highlighted in the prior proposal, water is the vital resource that fuels Alabama’s growth in agriculture, industrial output and technology. It is an indispensible resource that warrants no less than a state of the art technology to safeguard its integrity and to ensure its continual availability in a useable and safe form for future generations to come. Alabama has a sizeable animal husbandry based economy and the proximity of large tracts of surface water to large farms gives rise to a major problem: potential bacterial pathogens contamination via farm wastewater runoff. A water system contaminated with microbial pathogens from wastewater will have severe repercussions on Alabama’s economic growth in terms of reputation and water reliability, and more importantly, it will jeopardize public health. One of such bacterial pathogens as identified previously is Escherichia coli (E. coli) O157:H7. This organism has increasingly been implicated in waterborne outbreaks and is responsible for 73,000 illnesses, 2,100 hospitalizations and 60 deaths annually . The infective dose of E. coli O157:H7 is possibly fewer than 100 organisms . The symptoms usually appear within 2-4 days from the uptake of contaminated water. It often results in acute kidney failure without quick diagnosis and intensive care. Under the auspices of the previous grant, we have successfully embarked on the development of a rapid and accurate pathogen detection tool that is readily available and accessible for in-situ monitoring of the water bodies to support timely management and response. In this proposal, we will continue to build on our previous success in the preliminary demonstration of a viable technology for the rapid, accurate and robust in-situ detection of specific pathogens. We will focus on developing and demonstrating the capabilities of this technique, such as selectivity, sensitivity and resistance to inhibitor which will drive its eventual development into an enabling field tool and safeguard Alabama’s water resources. To keep the proposed work in line with the resources available through the 104 program, we will continue to concentrate on E. coli O157:H7 as the target pathogen. Other pathogens (e.g., Giardia lamblia, Legionella) can be subsequently added relatively easily after the base technology has been fully developed. Current technologies for pathogen detection and their limitations As described in the previous proposal, the traditional most probable number (MPN) method is limited by its lack of specificity and could not differentiate between benign and malignant species. Even with the recent improvement of biotechnology and genomics which has enabled new development in the DNA detection technology that is based on DNA amplification, amplification techniques such as real-time PCR (also known as qPCR) are still restricted to purified samples due to its inherent susceptibility to contamination. To use minimally prepared environmental samples in real-time PCR will result in either the amplification of undesired DNA (i.e., contaminants) along with the target DNA [3, 4] or the incomplete/failed amplification due to inhibitory mechanisms [5-10]. In order to avoid PCR inhibition due to contamination, field samples have to undergo extensive preparation in a laboratory environment in order to quantify the target DNA by real-time PCR. Furthermore, it is often required to check the amplicons against the DNA ladder via agarose gel electrophoresis to ensure that the target DNA has been amplified. Owing to its vulnerability to contamination, which is typical of gene amplification technique, the real-time PCR requires extensive steps and apparatus, including a gel electrophoresis, a gel imaging apparatus and a clean bench. Therefore PCR based techniques in its current state may not be ideal for further development into an in-situ capable technique. Research needs required for developing new in-situ capable pathogen detection technique Proper stewardship of Alabama’s vast water resources relies on the amount of data and tools available to formulate and execute effective management strategies. In other words, the availability of a miniaturized in-situ pathogen detection system will have an enormous impact on the way we manage the contamination of our water resources. It will open up numerous possibilities in terms of monitoring, tracing and rectifying the contamination source. However the development of such an in-situ pathogen detection system is contingent on the availability of a rapid, accurate, and economic detection technology. For this reason, we are committed to develop a rapid, accurate, in situ technique for the detection of pathogens in water at levels as low as 100 organisms per mL. The detection of our technique is based on the novel use of both fluorescent and magnetic nanoparticles which will be specifically assembled together only in the presence of the target pathogen DNA. This technique, unlike PCR, must be able to maintain its selectivity in the presence of other contaminants. Its sensitivity should also be comparable to that of PCR such that it can detect minimum infectious doses of about 100 organisms per mL. A short analysis time from sample injection to pathogen quantification is also critical for efficient real-time monitoring. In other words, there must be minimal processing steps from sample injection to quantification. The reagents used in the technique must be stable over a range of temperature for an extended time in order for it to be viable for in-situ use. Most importantly, the technique must be able to lend itself readily to the detection of multiple pathogens such as Giardia, Legionella and Campylobacter. In terms of economic consideration, this technique should use only a small quantity of low cost reagents and disposables in order to limit the cost per test to a few dollars. The research proposed here will specifically allow us to further develop a technique that will fulfill the above requirements. Capabilities of an ideal in-situ pathogen detection technology The ideal technique for in-situ bacterial monitoring must be able to maintain (1) its selectivity in the presence of other phylogenetically similar bacteria. (2) Its sensitivity should be also comparable or better than that of real-time PCR such that it can detect the minimum infectious doses, which is 100 organisms per mL of water for the case of E. coli O157:H7 [11, 12]. (3) The technique should be viable for ambient temperature incubation and should require minimum mechanical agitation during reaction. This will reduce the complexity of the apparatus required for using the technique outside a laboratory setting. (4) A relatively short analysis time (e.g., hours to minutes) from sample injection to pathogen quantification is also critical for effective monitoring. In other words, there should be minimal processing steps from sample injection to quantification. (5) The reagents used in the technique must be stable over a range of temperatures for extended time in order to prepare them in advance and to avoid reagent preparation insitu at the sampling site. (6) Most importantly, the technique should be resistant to a number of contaminants and inhibitors (e.g., humic acids, cations) [13-15] that are present in large amount in environmental samples. Additional interference to the reaction also includes residual reagents (e.g., surfactant, ethanol) from nucleic acid extraction. These inhibitors are well known to be detrimental to the amplification based quantification assay [8, 16]. Statement of the results, benefits, and/or information: Expected results and envisioned future work We will develop a rapid, accurate, modern yet robust, in-situ technique for the detection of E. coli O157:H7 in water at levels as low as 100 pathogenic organisms per mL. The basis of our technique is the novel use of advanced multi-functional nanoparticles which can be specifically assembled together only in the presence of the target pathogen. It can be easily extended to the detection of other pathogens once the core technology has been developed. Specific results of this research will be in the form of design and performance parameters investigated and established via a series of laboratory experiments. These parameters will include (1) sample size, (2) amount of reagents, (3) number of process steps such as incubation or mixing and their respective duration, and (4) detection sensitivity and specificity required. Parameters identified to be incompatible with existing instrumentations will be singled out for further investigation and innovation. For example in order to increase the sensitivity of the technique without demanding for a high performance spectrometer (detector), the number of fluorescent nanoparticles attached to magnetic beads (particle carrier) can instead be increased. This will result in the corresponding increase in the fluorescence intensity and therefore sensitivity. These studies will be contingent on the findings from the research proposed in this proposal. The results from this study will also define the instrumentation requirements for an in-situ system in a follow-up project. The purpose of the follow-up project is to pilot test the proposed technology in the field as well as to expand the detection to other pathogens. In the absence of appropriate commerciallyoff- the-shelf components, the technical parameters established in this research will define the performance specifications for future components development. For example, in order to miniaturize the spectrometer for in-situ instrumentation, a charge-coupled device (CCD) and light emitting diode (LED) may be used as a detector and a light source, respectively. The performance specifications of the CCD and LED will in turn be defined by the established technique parameters (i.e., assay sensitivity and sample size). Finally, the results of this research will lead to a technology that can be patented and commercially developed for public good. As such, this proposal represents a low-risk, high-return-to-society opportunity to significantly leverage the resources of the 104 program, while at the same time supporting graduate education and junior faculty development. Benefits of the in-situ pathogen detection system The successful development of the in-situ pathogen detection system will usher in a new paradigm in water resource management. This new paradigm shift will consist of two aspects: (1) Ability to conduct focused monitoring (e.g., after an outbreak) with increased temporal and spatial resolution and (2) Enhanced capability of routine monitoring. With the in-situ pathogen detection system, a wide range of monitoring can be carried out at multiple sites with unprecedented temporal and spatial resolution. For example, we will be able to perform high resolution studies on the timeline of pathogen contamination from their sources to human exposure. These real-time data points will allow us to formulate effective solutions such as the optimal locations of routine water sampling points as well as managing the source of contamination, which can include relocation. The in-situ system will also allow us to evaluate the effectiveness of existing management and clean-up strategies as well as to uncover previously undetected contamination pathways. For regular pathogen monitoring in water, the in-situ pathogen detection system will enable faster response time to trace the source of contamination in the event of an outbreak. For example, park rangers and water officials will be able to perform routine monitoring without the time-consuming need to collect samples and sending them to laboratories for analysis. Minimal laboratory expertise will be required to operate the developed in-situ system. With continuous improvement to the system driven by future funding from NIH, NSF, AWWA, WERF or EPA, it will eventually be autonomous without the need of a human operator. Multiple units of the autonomous system can be positioned in the field for ubiquitous monitoring of water pathogens via sensor networks. A real-time, spatially and temporally distributed water quality map will be an invaluable resource to both prevent and control pathogenic outbreaks and their costly aftermath in terms of human lives and resources.
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