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Development of PCR-Microplate Array for Salmonella Serovars and Specific Identification of Foodborne Pathogens Using Nanoparticles-Based Bio

Yehualaeshet, Teshome; Reddy, P. Gopal; Abdela, Woubit; Samuel, Temesgen; Kim, Moonil
Tuskegee University
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The goal of this research is to develop PCR-microplate array for the identification of different Salmonella serovars involved in outbreaks in the past few years and to develop nanoparticle-based pathogen detection from different types of food matrices.

The specific objectives are: 1) development of PCR-microplate array for rapid identification of Salmonella serovars. Although we were able to amplify pathogen DNA from food matrix, minimum pathogen detection using specific PCR amplification was more challenging in complicated food matrices, leading to specific objectives 2 & 3.
2) Development of DNA barcode immune-magnetic separation using target specific DNA and or pathogen specific antibody to concentrate minimally present pathogen followed by PCR amplification of the target sequences,
3) use of Au-nanoparticle aggregation assay and PCR as means of naked eye pathogen detection. Year one of the project will be dedicated to material acquisition, equipment purchase, target specific localization, design and modification of oligomers; year two of the project dedicated to further material and equipment acquisitions, specific target localization from Salmonella serovars, validation of Salmonella specific primers using conventional and real time PCR assays, customization of PCR-microplate array, and development of functionalized gold and magnetic nanoparticles. Finally, year 3 of this project will fully be involved in validation of the gold and magnetic nanoparticles in Salmonella inoculated meat, milk and fresh produce. This last year will also be dedicated to writing manuscripts and reports. Initial success will be determined by the ability of the test to specifically identify the pathogen DNA under question. In addition we anticipate detecting the smallest amount of pathogen present in different types of food matrices either with PCR or naked eye using the Au and magnetic nanoparticles. In order to attain these research objectives, one of the major activities includes mentoring of two graduate students participating in the different activities of this research project.

This project also creates food safety related research awareness to our professional veterinary students by involving them in summer research from the College of Veterinary Medicine Nursing and Allied Health, Tuskegee University. These students take part in the research activities and present their findings in the annually held Biomedical Research Symposium and the Veterinary Medical Symposium at Tuskegee University. The summer research program also includes undergraduate students from NIH/MBRS/MARC scholarship to take part in these research activities and present their findings in Annual Biomedical Research Conference for Minority Students. The expected outcomes are rapid and sensitive assays for the detection of foodborne pathogens in different food matrices. The developed assays would be useful for both agencies concerned with food safety issues and also food processing industries. In addition intellectual property protection and US patent applications will be submitted for the different diagnostic tools developed from this project.

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Non-Technical Summary:
Salmonella is one of the most notorious organisms because it is difficult to control infections attributed to this species. Multiple outbreaks due to Salmonella infections occur every year in the USA, and causes serious health and economical issues. Salmonella can be found in many different types of foods such as meats, eggs, fruits, vegetables, and even processed foods such as peanut butter. Contamination by Salmonella can occur anywhere from field to the kitchen. Outbreak by Salmonella has not declined in the past fifteen years, and even worse, every year different Salmonella serotypes have emerged as causative agents of foodborne illnesses. No single molecular diagnostic test is so far available to detect multiple foodborne Salmonella serotypes simultaneously. This project proposes three objectives 1) Develop simultaneous, rapid and robust identification tool for multiple Salmonella serotypes, 2) Develop nano-particle based specific identification of a small number of Salmonella from different food products, 3) Exploits the intrinsic nature of gold nano-particle aggregation to develop naked eye pathogen detection technology. The project involves different interdisciplinary approaches including microbiology, food safety, genomics and nanotechnology. Findings of this study will ensure emplacing an effective means of detection prior to any incident to identify and apply appropriate risk mitigation measures. Moreover, the output of this project could provide reliable detection techniques to identify pathogenic Salmonella in human food supply systems, before the agents reach the consumer. Rapid assays will be patented for commercial use, and findings will be disseminated in the form of publications and presentations. This project not only increases the research capacity of the lab, but it also provide an opportunity to minority students to participate in food safety and food security research by introducing them to advanced molecular and nanotechnology approaches.

The initial phase of the project mainly involves text mining, genome sequence mining and multiple and double alignments of different target gene sequences of Salmonella serovars in search for specific target motifs. These motifs will then be analyzed using in-silico PCR, and all available genome sequence databases for Salmonella enterica subsp. enterica non-specific binding and in-silico amplifications. Only motifs binding 100% with the target and showing limited or absence of hybridization to non-targeted serovars will be selected and used for further in-vitro validation using conventional and then real time PCR assays. Likewise, the result of the real time PCR will be interpreted by the presence of single amplification plot from the target Salmonella and none from the closely related Salmonella serovars. Once individual primers are validated for inclusivity and exclusivity they will be used to develop the PCR-microplate array for simultaneous identification of multiple Salmonella serovars. Direct identification of pathogen from any food matrix is a challenge that necessitates objectives 2 and 3 of this project. Well-validated target probes will further be used for surface modifications of Au and magnetic nanoparticles. First approach involves DNA barcode assay, for highly sensitive and specific detection of pathogen in food matrix, this aspect is unique as sensitivity of the DNA barcode amplification will be extremely high even in the presence of a single pathogen in the food matrix.
The second approach utilizes functionalized Au-NPs use for naked eye detection of minimum pathogen in food matrix. At the end of this project period students and faculty will acquire enough knowledge to optimize and validate functional nanoparticles. The output will be evaluated quantitatively by the new research infrastructure development and implementation to achieve the objectives of this project, the number of peer-reviewed publications and presentations by the PI's and graduate students, successful recruitment and graduation of two graduate students, and by the number of team oriented research proposals submitted for funding from programs such as USDA/AFRI Food Safety Research Initiative.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Food Defense and Integrity
Bacterial Pathogens
Risk Assessment, Management, and Communication
Nuts, Seeds