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<p>The objective of this nanotechnology-based project is to enhance food safety through the development of highly sensitive and specific nanofluidic systems targeting single pathogenic bacterial cells. The proposal will address</p>
<p>1) the design, fabrication and functional confirmation of nanofluidic reactor arrays,</p>
<p>2) the design and validation of fluorescent probes and PCR primers, and</p>
<p>3) the detection of pathogenic bacterial cells with nanofluidic digital PCR arrays.</p>
<p>The proposed nanofluidic systems will provide unprecedented sensitivity with minimized enrichment time (up to six hours) prior to analysis. Hence, the entire detection process could be accomplished within less than eight hours. The applications of small volumes will be organized in experiments with very-large-scale-integrated (VLSI) nanofluidic devices to target Campylobacter jejuni and Listeria monocytogenes cells. The long-term goal is to accomplish bacterial detection with a detection limit of less than 10 CFU/ml on nanoliter fluidic arrays within eight hours. The final product will be a new methodology of using nanoliter fluidic arrays for the detection of foodborne pathogens.</p>
<p>NON-TECHNICAL SUMMARY:<br/> There have been advances in the development of rapid methods for isolation and identification of foodborne pathogens. Conventional isolation methods relied on the use of traditional microbiology media, but unfortunately these methods are labor-intensive, time-consuming and, in many cases, require several days for completion. Hence, a rapid, reliable detection method of foodborne bacterial cells is important to improve food safety. Here, we propose a novel, nanotechnology-based method to enhance food safety by the use of highly sensitive and specific nanofluidic systems targeting single pathogenic bacterial cell detection through a unique and novel sample handling method. If the proposed work is successfully carried out, we could detect single cell level pathogenic bacterial cells without any enrichment steps, providing ultimate sensitivity.
<p>APPROACH:<br/>
Nanoliter or picoliter volumes of fluidic reactor arrays will be applied for the rapid and sensitive detection and identification of C. jejuni and L. monocytogenes cells collected from broiler meat samples. The nanoliter/picoliter scale fluidic array consists of a number of microvalves and individual addressable or integrated nanoreactors for amplifying target genes. These valves allow for the manipulation of the fluidic flow for delivering target pathogenic bacterial cells suspended in a PCR mixture to each nanoreactor. Efforts for designing, developing, and confirming molecular probes working in the principles of Fluorescent Resonance Energy Transfer (FRET) will be pursued to visualize the presence of target pathogenic bacterial cells. Different marker genes from Campylobacter isolates (including cadF, ceuE, hipO, etc.), and L. monocytogenes (hly and
inlB) isolates will be investigated. The ability to analyze the fluorescent intensity of the high-density nanoreactor array is also important for the identification of target bacterial cells through specific marker gene detection, and an image analysis system and software will be used.
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<p>PROGRESS:
<br/>2008/12 TO 2012/11
<br/>Target Audience: The target audiences of the funded project are nanobiotechnology related researchers and students who are participating active research and development of new technological tools for food safety. <br/>Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Six (6) graduate students and two (2) postdoctoral scholars have been supported and trained for food-safety related nanobiotechnolgy through this project. How have the results been disseminated to communities of interest? The developed system and methodology have been presented to food-safety related audiences through ample opporunities. The results were introduced to the potential end-users as asensitive and reliable way of detecting pathogenic bacteria wihotut over-night cultivation. What do you plan to
do during the next reporting period to accomplish the goals? Nothing Reported
<p>PROGRESS:<br/>
<br/>2009/12/01 TO 2010/11/30
<br/>OUTPUTS: A. Activities 1. Summary: We demonstrated a new method for the reliable detection and enumeration of food-borne pathogenic bacteria with the sensitivity of a single template in two hours by using microfluidic digital PCR 2. Primer/probe design and evaluation: We designed seven combinations of primer sets and probes targeting the hemolysin gene (hly) of L. monocytogenes and evaluated the specificity of primer sets and the functionality of probes through off-chip PCR experiments. The samples of genomic DNA purified from L. monocytogenes, L. innocua, L. grayi, L. seeligeri, L. welshimeri, and L. ivanovii were used for the initial materials of PCR. From the off-chip PCR and gel electrophoresis, we observed four combinations of primer sets and a probe successfully amplified the target genomic DNA of L. monocytogenes, and
showed an increase of fluorescence intensity as a function of PCR cycles. Finally, the combination A1, which has a maximum signal to background ratio, was selected through the quantitative assessment of reaction performances, and was for the microfluidic digital PCR to detect the genomic DNA and heat inactivated cell of L. monocytogenes with the sensitivity of a single template hereafter. 3. Microfluidic digital PCR: We detected and enumerated the food-borne pathogen L. monocytogenes by using an array of nanoliter reactors as a platform for detection. A series of template concentrations determined by the off-chip experiment were introduced into each panel of the reactor array, and microfluidic digital PCR was carried out. The time of reaction took less than 2 hours to give results. From the result, we clearly observed that microfluidic digital PCR is capable of the detection of
food-borne pathogenic bacterium with the sensitivity of a single template or cell. The efficiencies were 95.9% for genomic DNA and 79.9% for heat inactivated cells. Through the presented research, we demonstrated the reliable detection of L. monocytogenes with the sensitivity of a single template, which had been accomplished only by colony counting method.
<br/>PARTICIPANTS: Not relevant to this project.
<br/>TARGET AUDIENCES: Not relevant to this project.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.</p>
<p>PROGRESS:
<br/>2008/12/01 TO 2009/11/30
<br/>OUTPUTS: Activities 1. Summary: In this year, we have designed and fabricated a nanoliter reactor array chip and developed four combinations of a primer set and a probe to detect pathogenic bacteria, L. monocytogenes. 2. Primer/probe design and evaluation: We designed seven combinations of primer sets and probes targeting the hemolysin gene (hly) of L. monocytogenes and evaluated the specificity of primer sets and the functionality of probes through off-chip PCR experiments. The samples of genomic DNA purified from L. monocytogenes, L. innocua, L. grayi, L. seeligeri, L. welshimeri, and L. ivanovii were used for the initial materials of PCR. From the off-chip PCR and gel electrophoresis, we observed four combinations of primer sets and a probe successfully amplified the target genomic DNA of L. monocytogenes, and showed an
increase of fluorescence intensity as a function of PCR cycles. Finally, the combination A1, which has a maximum signal to background ratio, was selected through the quantitative assessment of reaction performances, and was for the microfluidic digital PCR to detect the genomic DNA and heat inactivated cell of L. monocytogenes with the sensitivity of a single template hereafter. 3. Design and fabrication of the nanofluidic chip: Several different nanoliter fluidic arrays have been designed by AutoCAD software and fabricated by using microfabrication techniques. A nanoliter fluidic array chip containing 10,000 nanoliter fluidic chambers with 10 different sample pads were successfully fabricated and their functionality was then confirmed. By using the fabricated chip, thermal reactions were tested and evaporation problems were found with nanoliter fluidic channels. Therefore, the filling
reagent for control channels has been changed to a polyethylene glycol (PEG) solution to provide for better thermal stability of the chip operation. It is clear that keeping thermal stability up to 95 C is extremely difficult with liquids in the order of 5 nanoliter volume of reagent because of the increased surface-to-volume ratio. Hence, efforts continue to achieve advanced chip design and fabrication and thermal reaction optimization for PCR. 4. Next year Plan: For the next year research, we will continue to evaluate the fluorescent probes and primers and perform microfluidic digital PCR with purified genomic DNA and cells of L. monocytogenes. Products: Newly designed primer sets and probes targeting L. monocytogenes: Four combinations of a primer set and a probe to detect L. monocytogenes were developed and their functionalities were confirmed. These combinations could be applied to
the sensitive and specific detection of L. monocytogenes by using microfluidic digital PCR hereafter. <br/>PARTICIPANTS: Not relevant to this project.
<br/>TARGET AUDIENCES: Not relevant to this project.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.</p>