Progress 12/01/08 to 11/30/12
Outputs
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. 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
Impacts
What was accomplished under these goals?
We developed a new detection method for food-borne pathogenic bacteria L. monocytogenes with detection sensitivity of a single template and in 2 hours of detection time. The method utilizes a microfluidic digital PCR, a method for amplifying the target gene in a nanoliter fluidic reactor, to achieve the sensitivity and the detection time. By using the method, we demonstrated the reliable detection of L. monocytogenes with the sensitivity of a single template, which had been accomplished only by the colony counting method. The colony counting method used widely for the detection of food-borne pathogens is the isolation of the organism by using a culture media. This method involves enrichment culture in selective media followed by a number of biochemical and serological tests for confirmation of the species. Although this method has the sensitivity of a single bacterium, there is a critical limitation because it takes 3 or 4 days to get results through the whole process. Alternative methods, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assays (ELISA), immuno-capture, and PCR-ELISA have reduced detection time to several hours or two days, depending on the extent of sample enrichment. However, achieving single cell level sensitivity has still remained challenging. Recently, real-time PCR technologies, which use capillary tubes or 96 well plates, have been widely introduced. The sensitivity required for single cell or single template detection still remains challenging because the reliability and reproducibility of real-time PCR is highly influenced by the sample processing methods. Therefore, instead of the current methods, the new method we developed could be a powerful tool for sensitive detection of various food-borne bacterial cells, as well as other pathogenic agents, such as influenza, cholera, malaria, etc.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
Single template detection of Listeria monocytogenes by microfluidic digital PCR
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Progress 12/01/09 to 11/30/10
Outputs
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. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We developed a new detection method for food-borne pathogenic bacteria L. monocytogenes with detection sensitivity of a single template and in 2 hours of detection time. The method utilizes a microfluidic digital PCR, a method for amplifying the target gene in a nanoliter fluidic reactor, to achieve the sensitivity and the detection time. By using the method, we demonstrated the reliable detection of L. monocytogenes with the sensitivity of a single template, which had been accomplished only by the colony counting method. The colony counting method used widely for the detection of food-borne pathogens is the isolation of the organism by using a culture media. This method involves enrichment culture in selective media followed by a number of biochemical and serological tests for confirmation of the species. Although this method has the sensitivity of a single bacterium, there is a critical limitation because it takes 3 or 4 days to get results through the whole process. Alternative methods, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assays (ELISA), immuno-capture, and PCR-ELISA have reduced detection time to several hours or two days, depending on the extent of sample enrichment. However, achieving single cell level sensitivity has still remained challenging. Recently, real-time PCR technologies, which use capillary tubes or 96 well plates, have been widely introduced. The sensitivity required for single cell or single template detection still remains challenging because the reliability and reproducibility of real-time PCR is highly influenced by the sample processing methods. Therefore, instead of the current methods, the new method we developed could be a powerful tool for sensitive detection of various food-borne bacterial cells, as well as other pathogenic agents, such as influenza, cholera, malaria, etc.
Publications
- Jong Wook Hong and Jae Young Yun (2010) Primers and probes for Listeria monocytogenes, U.S. Provisional Patent Application No. 61/310,566
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Progress 12/01/08 to 11/30/09
Outputs
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. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We developed a nanofluidic chip with 10,000 nanoliter reactor array and four combinations of a primer set and a probe to detect pathogenic bacteria L. monocytogenes with the detection sensitivity of a single template. The principle we proposed method to achieve the sensitivity is the amplification of a target gene in a nanoliter fluidic reactor. By using the method, we could achieve the reliable detection of L. monocytogenes with the sensitivity of a single template, which had been accomplished only by the colony counting method. The colony counting method used widely for the detection of food-borne pathogens is the isolation of the organism by using a culture media. This method involves enrichment culture in selective media followed by a number of biochemical and serological tests for confirmation of the species. Although this method has the sensitivity of a single bacterium, there is a critical limitation because it takes 3 or 4 days to get results through the whole process. Alternative methods, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assays (ELISA), immuno-capture, and PCR-ELISA have reduced detection time to several hours or two days, depending on the extent of sample enrichment. However, achieving single cell level sensitivity has still remained challenging. Recently, real-time PCR technologies, which use capillary tubes or 96 well plates, have been widely introduced. The sensitivity required for single cell or single template detection still remains challenging because the reliability and reproducibility of real-time PCR is highly influenced by the sample processing methods. Therefore, if the principle works successfully, the method could be a powerful tool for sensitive detection of various food-borne bacterial cells, as well as other pathogenic agents, such as influenza, cholera, malaria, etc.
Publications
- No publications reported this period
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