Item No. 1 of 1

INVESTIGATOR: Abdela, W.; Kim, M.; Samuel, T.; Yehualaeshet, T.; Reddy, G.

PERFORMING INSTITUTION:
TUSKEGEE UNIVERSITY
TUSKEGEE, ALABAMA 36088

DEVELOPMENT OF PCR-MICROPLATE ARRAY FOR SALMONELLA SEROVARS AND SPECIFIC IDENTIFICATION OF FOODBORNE PATHOGENS USING NANOPARTICLES-BASED BIO

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.

OBJECTIVES: 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.

APPROACH: 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.

PROGRESS: 2012/09 TO 2017/08
Target Audience:Researchers, students, journalists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduation of two Masters students, training of two PhD candidates and training of one Postdoctoral student, all of which were minority students. One of the masters student has granted a full scholarship to continue on her PhD studies in Auburn University. How have the results been disseminated to communities of interest? These results were presented and featured at "Retaking the Field: The Case for a Surge in Agricultural Research." Click here to access this full report. http://supportagresearch.org/2057-2/ And on the recent edition of the National Geographic News, plate, please see the link below, under the title the "Cost of A Brilliant Idea" http://www.nationalgeographic.com/people-and-culture/food/the-plate/2016/07/fed eral-money-needed-to-protect-us-food- American Society of Microbiology 2015 Four peer-reviewed manuscripts will be published soon, three are under write-up, one is peer reviewed and resubmitted. A PhD student work is still underway, in the Nano-particle approach to concentrate pathogen from different food matrix. The outcomes of this previous proposal such as established nano-food safety laboratory, and knowledge in the PCR microplate array are key steps towards the foundation of this proposal. We are confident that knowledge obtained from this project will help advance the new project with a more organized approach. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2012/09 TO 2017/08
What was accomplished under these goals? Some of the major achievements of grant # 2012-38821-20056 include: · Establishment of a Nano-Food Safety laboratory in the Tuskegee University, College of Veterinary Medicine, Department of Pathobiology laboratories, since the procurement of this grant we have purchased different equipment and facilities to be able to work on nanotechnology application in food safety Developed a microplate PCR array kit able to identify 23 different Salmonella serovars simultaneously, reportedly involved in different food borne outbreaks in the past two decades. The result simply shows the high throughput nature of the PCR microplate assay developed in our lab, not only detects the presence of Salmonella in the sample but it also specifically identifies which serovar or serovars are present in multiple contaminations. So far there is no single tool present that is able to tell the presence of multiple Salmonella serovar contamination. From this work an intellectual property disclosure form has been filled and presented to Tuskegee University, IP committee for a patent application called "Genetic array for the detection of multiple Salmonella serovars as alternative to serotyping", and this is now submitted to University lawyers handling IP Validation of PCR kit on over 200 different organisms and over 300 different food samples obtained from retail supermarkets and kit has proved to be highly specific and sensitive, and readily detect Salmonella serovar prior to culturing, at the enrichment step.

PUBLICATIONS (not previously reported): 2012/09 TO 2017/08
Type: Journal Articles Status: Published Year Published: 2017 Citation: Research Paper Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions SAYMA AFROJ,1,2,3 KHALED ALDAHAMI,2 GOPAL REDDY,2 JEAN GUARD,4 ABIODUN ADESIYUN,5 TEMESGEN SAMUEL,2 AND WOUBIT ABDELA2* 1Department of Biology and 2Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama 36088, USA (ORCID: http://orcid.org/0000-0002-9262-5117 [W.A.]) 3Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849, USA 4U.S. Department of Agriculture, Agricultural Research Service, Athens, Georgia 30602, USA; and 5School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, St. Augustine, Trinidad and Tobago MS 17-133: Received 28 March 2017/Accepted 19 July 2017/Published Online 23 October 2017

PROGRESS: 2016/09/01 TO 2017/08/31
Target Audience: Nothing Reported Changes/Problems:This is not really a major change it is a dear need of an instrument we found out that we raally need to to purchase to overcome our challenge and finish the expected work successfully. One of the important problem we need to surmount in order to expeditiously execute the experiments within the requested time frame is the fact that we need a simple equipment which will help us track proper functionalization, conjugation and pathogen capture onsite. This is because the specimens in this work are very delicate, unstable and biologically hazardous to transport to the closest research facility where we may be able to do this, apart from the fact that doing so will hamper progress in the continuity of the entire chain process. This has being our major challenge, as we could not successfully track nanoparticle functionalization, conjugation and pathogen capture by any quantitative or qualitative means. Electron microscopy gave results, which is very difficult to tell if functionalization occurred. As a way of tracking the success of the process, we discovered that the Zetasizer nano ZSP is very in tracking the success and progress of the work after we requested a demonstration equipment to explore its usefulness in this project. We realized that this equipment which is sold with a customized computer can quickly measure changes in surface electrical properties and sizes of the nanoparticles as they are functionalized, conjugated and used as biosensors to capture pathogens in situ, and gives quantitative results which is easy to interpreted, conclusion and immediately redefine a line of action if needed, unlike the time consuming electron microscopy which we have relied on from the beginning. Though electron microscopy is needed, it is an occasional supporting tool after confirmation of proper functionalization with the Zetasizernano equipment. Because it is more expensive, time consuming and difficult to tell if functionalization has occurred with micrographs of the specimen. This gets more difficult as the specimen becomes more complex with functionalization, conjugation and pathogens. Since, the cost of the Zetasizernano ZSP and its accessories are well within the budget, we sincerely request your approval to purchase one to help us in charactering our nanosensors from the remaining funds and successfully complete our project in the coming calendar year. As a result I need a permission to buy Zetasizernano ZSP with $44,000 to successfully complete the project work. What opportunities for training and professional development has the project provided?-Two masters student graduated - PhD student is still working on the project, - We planned to take one more minority Masters student in the coming academic year How have the results been disseminated to communities of interest? The first manuscript is published in the Journal of Food protection and will available to public in the coming edition Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions SAYMA AFROJ,1,2,3 KHALED ALDAHAMI,2 GOPAL REDDY,2 JEAN GUARD,4 ABIODUN ADESIYUN,5TEMESGEN SAMUEL,2 AND WOUBIT ABDELA2* The patent is also submitted and it is in lawyers office, "Title of the patent is Genetic array for the detection of multiple Salmonella serovars as alternative to serotyping" Inventors: Woubit Abdela, Khaled Aldhami, Sayma Afroj, Temesgen Samuel and Gopal Reddy Our PhD Student won first Prize in her category of Poster competition at the Microscopy and Microanalysis Conference at Saint Louis MO, August 6-10 for her research entitled Nanomaterial-based receptor conjugates for capture and rapid detection of Salmonella Enteritidis Kiara Cousin1, Boniface Tiimob2, David Baah3, Ceasar Fermin1, Temesgen Samuel1, Woubit Abdela1 What do you plan to do during the next reporting period to accomplish the goals?Activities for the requested no-cost extension period (9/1/2017 - 8/31/2018) Continue to work with PhD student in the nanoparticle assay, and colorimetric detection Optimize the final DNA barcode immune-magnetic separation assay using newly developed Salmonella targets, to allow pathogen DNA concentration from mixed samples such as different food matrices, using the help of the new instrument we plan to purchase and expand our capacity of working with nanoparticles in College of Veterinary Medicine research facility. Finalizing the last two manuscripts under preparation, Working with one more new minority MSc student to expand the microplate array with additional seven serovars Closing the Project.

IMPACT: 2016/09/01 TO 2017/08/31
What was accomplished under these goals? - Microplate has been designed and submitted for patent as of now to specifically genotype 23 Salmonella serovars wth the extension of 7 more serotypes with the goatl of 30 serovar genotyping microplate PCR array, -A nano food safety lab has been established with all materials needed with the exception of Zeta potential which we plan to obtain in the coing last year. -We are still in the process of Optimizing our naked eye and DNA barcode detection assays

PUBLICATIONS: 2016/09/01 TO 2017/08/31
Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions SAYMA AFROJ,1,2,3 KHALED ALDAHAMI,2 GOPAL REDDY,2 JEAN GUARD,4 ABIODUN ADESIYUN,5 TEMESGEN SAMUEL,2 AND WOUBIT ABDELA2*

PROGRESS: 2015/09/01 TO 2016/08/31
Target Audience: Nothing Reported Changes/Problems:Major changes were described in Pharagraph 1 above Breifly, modification of procedure we used as stipulated on the proposal and developing of a new approach using peptides from Salmonella specific phages as capturing agents. We are still hoping to modify the protocol to use our specific primers as capturing agent. -The second major bottleneck in the past year was the departure of our trained PhD student. He found a job in USDA, CA so has dropped out of the program, we now have a new PhD student training to do this last part of the project as her dessertation proposal What opportunities for training and professional development has the project provided?Bottleneck in the experimental stages of DNA barcode assays, and other issues -The initial step of using the newly developed primer targets, as a capture agent of Salmonella did not produce substantial results using our previously proposed procedure. We used our protocol that includes the thiol-modified oligonucleotides and biotinylated probe DNA will be immobilized onto the AuNPs (13 nm in diameter) and the streptavidin-labelled magnetic beads, respectively. Following the lysis of pathogen-contaminated food matrix, the magnetic beads and AuNPs will be mixed with the lysed samples. The magnetic beads-AuNPs complex is formed through the target DNA in the presence of the specific pathogen in food matrix, magnet-separated, and subsequently PCR (Polymerase Chain Reaction)-amplified. So this required modification and developing of a new approach using peptides from Salmonella specific phages as capturing agents. We are still hoping to modify the protocol to use our specific primers as capturing agent. We discussed with our collaborators in Michigan State University, the plan will be to use their protocol and adopt our targets and rerun the experiments in the coming academic year, and rerun some of the experiments in their laboratory. -The second major bottleneck in the past year was the departure of our trained PhD student. He found a job in USDA, CA so has dropped out of the program 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,-Underway Use of Au-nanoparticle aggregation assay for Salmonella detection from different food samples-Underway Manuscript submissions-Yes Publication and patent fees of result finding and developed microplate array-Not Accomplished Yet Laboratory consumables, equipment and office supplies to my PhD student working in this part of the proposal-Yes Purchasing of actual food samples to test the developed tools in activities 1 and 2 above-Yes How have the results been disseminated to communities of interest?These results were presented and featured at "Retaking the Field: The Case for a Surge in Agricultural Research." Click here to access this full report. http://supportagresearch.org/2057-2/ And on the recent edition of the National Geographic News, plate, please see the link below. http://www.nationalgeographic.com/people-and-culture/food/the-plate/2016/07/fed eral-money-needed-to-protect-us-food-supply/ What do you plan to do during the next reporting period to accomplish the goals? Recruiting a new PhD or Masters student able to work on Magnetic nanoparticle and DNA barcode assay. 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, Use of Au-nanoparticle aggregation assay for Salmonella detection from different food samples Manuscript submissions after filing a patent Publication and patent fees of result finding and developed microplate array, with a plan to expand the microplate array with additional seven serovars

IMPACT: 2015/09/01 TO 2016/08/31
What was accomplished under these goals? In the last years reporting period, we planned to accomplish testing of our microplate array in actual food obtained from retail supermarkets, for that we have purchased about 200 different food samples, that include fresh produce, pork, ground beef, chicken meat (Chicken thigh and chicken neck) Sample size of different food purchased for testing the presence of Salmonella Fresh Produce Pork Ground beef Chicken thigh Chicken neck Tested Positive Tested Positive Tested Positive Tested Positive Tested Positive 40 0 40 0 40 5 40 1 40 32 RV broth extracted total DNA subjected to our newly developed Salmonella serovar PCR-Microplate array Serotypes identified Ground beef Chicken thigh Chicken neck No % No % No % S. Montevideo 5 12.25 - - S. Kentucky - 0 1 2.5 32 80 S. Paratyphi C 1 2.5 1 2.5 1 2.5 S. Newport - 0 1 2.5 1 2.5 S. Agona - 0 - 0 - 0 S. Arizoniae 1 2.5 1 2.5 2 5 S. Choleraesuis - 0 1 2.5 4 10 S. Heidelberg - 0 1 2.5 21 52.5 S. Diarizonae 1 2.5 1 2.5 4 10 S. Johannesburg - 0 - 0 1 2.5 S. Typhi - 0 1 2.5 - 0 S. Typhimurium - 0 - 0 1 2.5 S. Schwarzengrund - 0 - 0 3 7.5 S. Enteritidis - 0 - 0 2 5 Isolates from each of the above RV broth were send to USDA lab in Athens GA for further isolation, the lab uses traditional serology assay combined with serotyping by sequencing of a region called intergenic sequence ribotyping (ISR). Here is the final report, summarized in the next table. Salmonella isolates by serological identification, PCR and sequencing ISR Serotypes isolated Ground beef Chicken thigh Chicken neck S. Montevideo UN0033 5 - - S. Kentucky8,20;-;z6 - 1 32 The result simply shows the high throughput nature of the PCR microplate assay developed in our lab, not only detects the presence of salmonella in the sample but it also specifically identifies which serovar or serovars are present in multiple contaminations. So far there is no single tool present that is able to tell the presence of multiple Salmonella serovar contamination. (Please compare the above two tables). Additionally we transferred this technology for on-site Salmonella detection tool, using the newly developed targets for LAMP assay, and the result is promising as we have proved these targets easily adopted for on-site detection of multiple, at least 8 Salmonella serovar contamination.

PUBLICATIONS: 2015/09/01 TO 2016/08/31
Type: Journal Articles Status: Under Review Year Published: 2017 Citation: NA

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:Target Audiences were students from Tuskegee Univesity campus, scientists and students from all over the world at International meetings of American Society of Microbiology, ASM2015 and Alabama Academy of Science (AAS2015), Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?- Two masters students were graduated and their Thesis were submitted to Graduated School of Tuskegeee University - A PhD student work is still underway How have the results been disseminated to communities of interest?Presentation at American Society of Microbiology, ASM2015 and Alabama Academy of Science (AAS2015), presented by both masters' students where both were awarded a travel grant. Following are their abstracts for ASM and AAS respectively Development of real time PCR using novel genomic target for multiple Salmonella serovars detection from milk SaymaAfroj1,2, Khaled Aldahami2, Abiodun Adesiyun3, Temesgen Samuel2 and Woubit Abdela2 1Department of Biology, Tuskegee University, Tuskegee, AL-36088, 2College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL 36088; 3School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, St. Augustine, Trinidad and Tobago Abstract In our study we selected five Salmonella serovars based on their outbreak causing capacity to develop highly sensitive and specific novel genomic and plasmid target based PCR. The serovars include S. Heidelberg, S. Dublin, S.Hadar, S. Kentucky and S.Enteritidis. Through extensive genome mining of protein databases of these serovars and comparative genomic analysis, we identified unique genome loci, which we utilized to develop a conventional and real-time detection platform. Most of the proteins we identified were hypothetical or putative whose function is not clear yet. We used V-NTI Advanced-11 for the design and modification of primers. The designed primers were validated by in-silico PCR, and further BLASTed using NCBI and PATRIC databases. Initial validation of inclusivity and exclusivity assay involved testing of these primers with the genomic DNA of corresponding Salmonella serovars, 96 non-target Salmonella serovars, and ~30 closely related bacterial species, tested in-vitro by conventional PCR and real time PCR. The results obtained from both conventional and real-time PCR, indicated that our developed primers were highly specific with 100% inclusivity and 100% exclusivity. The limit of detection of these five serovars with DNA sensitivity was found to be 58.8fg/µl for S.Heidelberg, 42.2fg/µl for S.Kentucky, 200fg/µl for S.Hadar and 79.6fg/µl for S.Enteritidis, with corresponding 11.8, 8.4, 40, 16 bacteria cells/ml respectively. Likewise the initial multiplex assay performed in pooled DNA of the five serovars containing 266-533pg/µl in 30-15µl reaction volumes, provided a specific amplification of each of serovars. Sensitivity assay was also performed in artificially inoculated milk with pooled Salmonella serovars each mixed with 0.5OD at 301nm, and diluted 10 fold up to 10-8 containing 1 CFU. The result showed a detection limit of 1-10 CFU per 25 ml of milk samples. The multiplex assay from food will be further validated with Taqman assay containing internal amplification control. In conclusion our newly developed assay will meet the need for rapid detection and serovar identification in any food testing laboratories and processing plants foreseeing to deliver quality food products to consumers. Key words: Salmonella, Novel genomic target, Protein databases, in-silico PCR, NCBI and PATRIC databases, real-time PCR. Acknowledgement This research is supported by the USDA/AFRI/CBG Grant 2012-3882120056, we also would like to thank Auburn USDA Diagnostic laboratory and USDA diagnostic lab in Athens GA for providing the Salmonella serovars. Novel Genomic Target For Specific & Sensitive Identification Of Salmonella Serovars From Retail Meat Samples *Khaled Aldahami1*, Sayma Afroj 1,2, Abiodun Adesiyun3, Temesgen Samuel1, and Woubit Abdela1 1,2Tuskegee University, Tuskegee, AL 36088, 3University of the West Indies, St. Augustine, Trinidad and Tobago Genome sequences of Salmonella serovars were used to localize target proteins through BLASTp analysis. Corresponding target genes were retrieved to V-NTI Advanced-11 (Invitrogen, USA) for the design and modification of primers. Comprehensive analysis of these primers included tests for nonspecific intra- and inter-serovars binding or reaction with closely related bacteria; Tm, dimer and hair-loop formation were assessed using V-NTI. To date we have successfully designed primers from unique genome regions for 5 Salmonella serovars that are responsible for multi foodborne outbreaks. Prior to validation of individual primers using conventional and real time PCR assay, in-silico PCR was performed across the genome sequence of available Salmonella serovars. Conventional and real time PCR assay validations were performed by probing all of the primers against a wide range of Salmonella and closely related bacteria, reaching a 100% inclusivity and exclusivity. Sensitivity assays of the 5 pooled Salmonella serovars artificially inoculated in chicken meat, with each of which ~0.5 OD at 300nm resulted in a detection limit as low as 2 bacteria in 25 g of meat. Eventually, these primers will be aliquoted in PCR micro-plate arrays for rapid and simultaneous identification of multiple Salmonella serovars. Our finding plays a major role for rapid, highly specific and effective detection of this pathogen. The USDA/AFRI/CBG Grant 2012-3882120056 supported this research What do you plan to do during the next reporting period to accomplish the goals?Activities for the requested no-cost extension period (9/1/2015 - 8/31/2016) 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, Use of Au-nanoparticle aggregation assay for Salmonella detection from different food samples Manuscript submissions Publication and patent fees of result finding and developed microplate array Laboratory consumables, equipment and office supplies to my PhD student working in this part of the proposal Purchasing of actual food samples to test the developed tools in activities 1 and 2 above

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? In the last years reporting period, we planned to accomplish the following specific objectives: 1. Perform DNA sensitivity assay of each target specific PCR The result of the sensitivity assay from genomic DNA of the 5 Salmonella serovars showed different detection limit. For each serovar, every successful primer was tested for minimum DNA detection limit. The minimum detection limit was for Salmonella Agona 1F/1R primer, which was 44.2 fg/μl corresponding to 9 bacterial cells, whereas the highest detection limit was for Salmonella Bareilly 1F/1R 448 fg/μl corresponding to 90 bacteria cells. The limit of detections of the five serovars with DNA sensitivity were found to be 58.8 fg/µl for S.Heidelberg, 42.2 fg/µl for S.Kentucky, 200 fg/µl for S.Hadar, 63.4 fg/µl for S.Enteritidis and 26 pg/ µl for S.Dublin, which corresponded with11.8, 8.4, 40, 13, 5200 bacterial cells/ml respectively. The cut-off value for SYBR Green real time PCR was determined at the Ct value of 30 cycles. 2. Perform sensitivity assay of detecting Salmonella from intentionally contaminated food such as meat and milk The multiplex conventional assay was able to detect all Salmonella serovars from 10-1 to 10-6 dilution of the pooled sample that contained 100 CFU. At 10-7, dilution containing 1 - 10 CFU, the assay detected S. Hadar, S. Dublin, S. Enteritidis, S. Kentucky except S. Heidelberg. The individual PCR from 10-7 also showed negative amplification for S. Heidelberg, which confirmed the absence of this serovars at this dilution or the amount of DNA was so low that it was not measurable. The TaqMan Multiplex assay where serovars Heidelberg, Enteritidis and Dublin were targeted was able to detect ALL OF those three serovars from 10-1 to 10-6 dilution of the pool sample, which contains 100 CFU, however at 10-7 dilution containing 1 - 10 CFU, the assay was able to detect only two of the serovars but not S. Heidelberg. The interpretation of this observation is that both multiplex conventional and multiplex TaqMan assays were 100% in agreement. The cut-off value for TaqMan assay was determined at CT value of 35 cycles. 3. Optimize RT-multiplex PCR for simultaneous detection of food multiple Salmonella serovars The conventional multiplex assay was able to detect serovars Hadar (354 bp fragmant), Heidelberg (289 bp) and Dublin (118 bp) in Set1 at a concentration of 266 pg/µl and serovars Enteritidis (325bp) and Kentucky (246 bp) in Set2 at a concentration of 400 pg/µL. Individual serovars were also detected by individual primer sets at a concentration of 533 pg/µL of reaction volume. Same results were also obtained from artificially contaminated milk samples. The multiplex TaqMan assay was performed to determine the level of detectable DNA concentrations corresponding to the genomic equivalent (GE) of S. Heidelberg, S. Enteritidis and S. Dublin. The standard curve was generated using various concentrations the three serovars. For S. Heidelberg DNA from 30ng (6*106GE) to 15.36fg (3*100 GE) were used, for S. Dublin from 2.18ng (4.36*105 GE) to 1.1 fg (3*10-2GE) and for S. Enteritidis from 11ng (2.2*106 GE) to 5.6fg (1*100GE) were used. The slopes for the standard curve of S.Enteritidis on FAM, S. Heidelberg on ROX and S. Dublin on Cy5 were -3.460, -3.592 and -4.093 respectively. The regression curves were generated for these three serovars based on the varying amounts of bacterial DNA. A good linearity response was shown for each of the standard curves of the serovars. The R2 value was 1.000 for S. Enteritidis, 0.998 for S. Heidelberg and 0.992 for S. Dublin. The results indicated that the multiplex real-time PCR successfully detected the minimum DNA, which was 75.8fg (1.53*101) for S. Heidelberg, 140.8fg (2.8*101) for S. Enteritidis, and 3.48 pg (6.96*102) for S. Dublin. PCR efficiencies calculated from the standard curve gave efficiency of 89.8% for S. Heidelberg; 94.5% for Enteritidis, and 75.5% for S. Dublin. 4. Detection of natural contamination of Salmonella from chicken meat samples Among the thirty chicken samples, twelve (40%) were positive by both cultural techniques by conventional PCR with Salmonella specific invA gene. Those twelve positive samples were tested by conventional PCR with the serovar specific primers individually. The result of serovar specific PCR showed six positive samples (20.0%) for serovar Kentucky (B1, B2, B3, C1, C2 and C3). Analysis using the S. Enteritidis specific PCR revealed seven positive samples (23.3%) for S. Enteritidis (B1, B2, B3, C1, C2, C3 and I2), the six samples (20.0%) (B1, B2, B3, C1, C2 and C3) gave double contamination with both S.Kentucky and S.Enteritidis. Using the TaqMan assay, targeting only S. Heidelberg, S. Enteritidis and S. Dublin, same results were obtained as in the conventional PCR for S. Enteritidis. However, this assay also amplified nine samples as S. Heidelberg; among them seven were concurrently amplified as serovar Enteritidis. The twelve positive samples were sent to USDA diagnostic laboratory, Athens, Georgia for serotyping and sequencing. They confirmed five samples (B1, B2, C1, C2 and C3) as S.Kentucky, which were also positive as Kentucky by the assay run in our lab. 5. Customize 96-well PCR microplate array for simultaneous detection of multiple Salmonella serovars The following layout was adopted for the customizable microplate Salmonella serovar PCR array based on the successfully designed and validated primers. A pool of DNA samples from 23 Salmonella serovars genomic DNA was used for the validation with = 1.3 µg/ µl per well per reaction 1 2 3 4 5 6 7 8 9 10 11 a SP1 STm1 SAG1 SAZ1 SBD1 SBY1 SBUP1 SD1 SHD1 SSZ1 SMIS1 b SP1 STm1 SAG1 SAZ1 SBD1 SBY1 SBUP1 SD1 SHD1 SSZ1 SMIS1 c SN1 SSP1 SAG2 SAZ2 SBD2 SBY2 SBUP2 SD2 SHD2 SSZ2 SMIS2 d SN1 SSP1 SAG3 SAZ2 SBD2 SBY2 SBUP2 SD2 SHD2 SSZ2 SMIS2 e SN2 ST1 SHAD1 SCH1 SKT1 SW1 SJG STS1 SDIZ1 SE1 SMV1 f SN2 ST1 SHAD1 SCH1 SKT1 SW1 SJG STS1 SDIZ1 SE1 SMV1 g S S S S S S S S S S S h P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 P1,P2,P3 ParatyphiC TYPHIMURIUM Agona-1F/1R Arizoniae-1F-m/1R-M Baildon-1F-m/1R-m Bareilly-1F-k/1R-K Braenderup-1F-m/1R-m Dublin-1F-p/1R-p Heidelberg-1F/1R Shwazengrund-1F-m/1R-m Mississipi-1F/1R Newport-1F-m/1R-M SAINPAL Agona-2F/2R Arizoniae-2F-m/2R-m Baildon-2F-m/2R-m Bareilly-2F-k/2R Braenderup-2F/2R Dublin 1F-1R s. Heidelberg 1F-1Rs Shwazengrund-2F-m/2R-m Mississipi-2F/2R Newport-1F-3/1R-3 TYPHI Hadar 1F/1R Choleraesuis-2F-m/2R-m Kentucky-2F/2R Wandsworth-1F/1R Johannesberg 2F-2R Thomson-2F/2R Diarizonae-1F/1R Enteritidis-all-2F/2R Montevideo-2F-m-2/2R-m-2 Chicken E1 Positive: Arizonae, Choleraesuis, Johannesberg, Heidelberg, Enteritidis Chicken I1: Positive: Salmonella serovars Typhimurium, Arizonae, Johannesburg, Schwarzengrund, If the PCR cycle is selected to 22, then only Typhimurium would be positive. 7. Submission of a first manuscript

PUBLICATIONS: 2014/09/01 TO 2015/08/31
No publications reported this period.

PROGRESS: 2013/09/01 TO 2014/08/31
Target Audience: -Presentation to University Audience -Presentation to Tuskegee Community during the Annual Biomedical Symposium Held every September -Seminars to MPH and MSPH sudents of the College of Veterinary medicine Nursing and Allied Health Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided training for two graduate students, whom have been closely working with this project in the past one year, and a PhD student who is involved in the Nanoparticle functionalizing part of the project, the PhD student will be intensively involved in the use of the above unique targets of multiple Salmonella serovars for functionlaization of Gold and Magnetic nanoparticles for eventual use in the DNA barcode assay. He is also looking at the possible use of Salmonella selective serovar phages as biomarkers of pathogen concentration in food. In Summary, 2 -Graduate and 1-PhD students are actively working How have the results been disseminated to communities of interest? Papers were presented in the Annual Biomedical Symposium at Tuskegee Univesriy Title of presentations during different occasions by two graduate students: 1= Development of real time PCR using Novel genomic Target for Multiple Salmonella serovars Detection from Milk 2= Novel genomic tools fr Specific and Real time Detection of Salmonella serovars What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we wish to accomplish at least submission of two manuscripts from Data generated by the Graduate students, with the addition of the following specific objectives: 1. Perform DNA sensitivity assay of each target specific PCR 2. Perform sensitivity assay of detecting Salmonella from intentionally contaminated food such as meat and milk 3. Optimize RT-multiplex PCR for simultaneous detection of food multiple Salmonella serovars 4. Customize 96-well PCR microplate array for simultaneous detection of multiple Salmonella serovars 5. Use Salmonella targets for the functionalization of gold and magnetic nanoparticles for specific concentration and higher sensitivity detection of Salmonella from food matrix,

IMPACT: 2013/09/01 TO 2014/08/31
What was accomplished under these goals? During this year two period of the project, we have sucessfully identified unique genome regions of 24 Salmonella serovars, and rigrously validated this targets to over 100 Salmonella serovars purchased from ATCC, NVSL Ames-Iowa and from USDA diagnostic labs of Auburn Al, and Athens, GA. The Validation includes, the details mentioned below, Genomic and expressed gene data mining Completed genome sequence data from most of the Salmonella serovars, including incomplete genome sequence for few of the Serovars were retrieved from the NCBI (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) microbial genome-sequencing database. Most recently published references of comparative genomics of Salmonella serovars were used in target region selection. A BLAST search was used in the selection of specific amino acid sequences showing >50% polymorphism and unique genomic regions of each Salmonella serovars were targeted for the design of primers. Specific primers were designed from genes and coding regions located uniquely to the Salmonella serovars of our interest. These primers were then analyzed in-silico for specific binding across the genome sequence of other Salmonella serovars. For more stringency assessment we used V-NTI Advanced-11 (Invitrogen, USA) for the design and modification of primers having a 3` end similarity with the closely related species. All available whole genome sequence and partial sequences from prokaryote genome database of NCBI were used for the in-silico validation of the specific primers. Comparative genomic analysis In a search for specific region a BLAST analysis was performed for all the serovars containing a coding sequence of > 100 aa, further COBALT alignment analysis was used to localize more specific regions for serovar under question. Once the specific amino acid sequence was localized we have used the nucleotide sequence of this sequence for further BLAST analysis on both PATRIC (PATRIC, the bacterial bioinformatics database and analysis resource) database and V-NTI 11.5 Advanced software. Regions of prominent variation were employed for the design of primers. Primer design and preliminary analysis These primers went through rigorous testing before the experimental validation. This included oligo-dimer, palindromes, repeats, 3`GC content, hair-loop formation and standardization of all primers to have similar melting temperature, which is one of the requirements for the simultaneous use of these primers. Primers fulfilling the required criteria further analyzed for possible binding to a different location within the same whole genome (intra-serovar) sequence using V-NTI motif search analysis tool. In-silico PCR In silico PCR validations were performed using both http://insilico.ehu.es/PCR/ and V-NTI, only primers giving specific amplifications were selected for further validation using conventional and real time PCR assay. Primers that showed the most specificity to the target molecule were ordered from Integrated DNA technologies (IDT, IA). In vitro PCR and analysis All PCR reactions were set up in an isolated PCR station (AirClean Systems, NC) that was UV-sanitized daily and after each use. Initial conventional PCR was performed to validate the specificity of the primers for respective Salmonella serovars. DNA from 100 Salmonella serovars representing major foodborne outbreaks and frequency of isolation were used for the validation (Please refer to Table 1). Single target PCR was performed in a 20µl final volume containing 10µM of forward and reverse primers, 12.5µl of Pwo Master mix containing 1.25U of Pwo enzyme, 2mM MgCl2 and 0.2mM dNTPs (Roche Diagnostics, Mannheim Germany). The PCR amplification profile for this initial assay consists of 10min at 95oC, followed by 30 cycles of 15seconds at 95oC, 15seconds at 60oC, and 15seconds at 72oC using Master cycler pro (Eppendorf, Humburg, Germany). Presences of single band were analyzed using gel electrophoresis. Real-time PCR Real time PCR assay was performed using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) for final validation & verification. A specific amplification was obtained with all the primers used to amplify the respective Salmonella serovars. A reaction volume of 20µl containing 500nM of forward and reverse primers, 10µl of 2X Brilliant III Ultra-Fast SYBER Green master mix, 0.3µl ROX reference dye, 1µl of DNA template, nuclease free H2O added to the final volume was used for the real-time PCR. Cycling conditions were consisted of one cycle of segment 1, 2min at 95oC; followed by 27 cycles of segment 2, 10seconds at 95oC, 30seconds at 60oC; completed by one melting curve cycle of 1min at 95oC, 30seconds at 65oC, and 30seconds at 95oC. The Names of the list of primers which will be used to coat a 96-well PCR microplate array after individual rigrous testing using methods mentioned above includes, Agona-1F Agona-1R Agona-2F Agona-2R Arizoniae-1F-m Arizoniae-1R-m Arizoniae-2F-m Arizoniae-2R-m Baildon-1F-m Baildon-1R-m Baildon-2F-m Baildon-2R-m Baildon-2R-m Bareilly-1F-k Bareilly-1R-k Bareilly-2F-k Bareilly-2R Braenderup-1F-m Braenderup-1R-m Braenderup-2F Braenderup-2R Choleraesuis-1F-m-K Choleraesuis-1R-m-K Choleraesuis-2F-m Choleraesuis-2R-m Diarizonae-1F Diarizonae-1R Dublin-1F-p Dublin-1R-p Hadar-1F Hadar-1R Hadar-2F-m Hadar-2R-m Heidelberg-1F Heidelberg-1R Heidelberg-2F-s Heidelberg-2R-s Johannesburg-2F Johannesburg-2R Kentucky-1F-Gam Kentucky-2F Kentucky-2R Kentucky-2R-Gam Montevideo-1F Montevideo-1R Montevideo-2F Montevideo-2F-m-2 Montevideo-2R Montevideo-2R-m-2 Newport-1F-m Newport-1R-m Newport-1R-3 Newport-1F-3 Paratyphi C-1F-s Paratyphi C-1R-s Shwazengrund-1F-m Shwazengrund-1R-m Shwazengrund-2F-m Shwazengrund-2R-m Tennes-1F Tennes-1R Tennes-2F Tennes-2R Wandsworth-1F Wandsworth-1R Enteritidis-all-2R Enteritidis-all-2F Mississipi-1F Mississipi-1R Mississipi-2R Mississipi-2F Thomson-2R Thomson-2F Typhimurium Saintpaul Typhi

PUBLICATIONS: 2013/09/01 TO 2014/08/31
Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Sayma Afroj,Khaled Aldahami, Abiodun Adesiyun, Temesgen Samuel and Woubit Abdela: Development of real time PCR using novel genomic targets for the detection of multiple Salmonella serovars from milk. September 2014, the 15th Annual Biomedical symposium, Tuskegee AL.

PROGRESS: 2012/09/01 TO 2013/08/31
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> During the Past one year the visit to MSU and ASU provided the PD with training opportunities to start up the project work. At the end of this first year two graduate students will fully involve with the project. How have the results been disseminated to communities of interest? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> There are no results generated during this period of the project. What do you plan to do during the next reporting period to accomplish the goals? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> The next reporting period fully focuses in the identification and validation of Salmonella serovar specific targets, at the same time graduate students will involve in the identification of different Salmonella serovars from different food sources to be used in the validation of the target primers. Validation involves in-silico across a wide array of Salmonella serovars and V-NTI motif search with in the genome of the same serovars and closely related bacterial species. At the end of this period we will have Salmonella serovars target specific primers ready to be validated in-vitro, for their ultimate utilization of custom microplate array. In the mean time the PI and graduate students functionalize and validate gold and magnetic nanoparticles for the specific capture of target Salmonella serovars from food components.

IMPACT: 2012/09/01 TO 2013/08/31
What was accomplished under these goals? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> The major accomplishments in this one-year period of this grant are mainly preparation of the working conditions for the successful execution of the project objectives. These steps include: 1) Establishment of Food Nanotechnology lab for the effective running of objectives, 2) Collaborative laboratory Visit of the Nano-biosensor laboratory in Michigan State University, and Center for Nano-Biotechnology Research at Alabama State University (ASU); and 3) Retrieval and collection of so far sequenced Salmonella serovars into our genome Database software V-NTI

PUBLICATIONS: 2012/09/01 TO 2013/08/31
No publications reported this period.

PROGRESS: 2012/09/01 TO 2013/08/31
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> During the Past one year the visit to MSU and ASU provided the PD with training opportunities to start up the project work. At the end of this first year two graduate students will fully involve with the project. How have the results been disseminated to communities of interest? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> There are no results generated during this period of the project. What do you plan to do during the next reporting period to accomplish the goals? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> The next reporting period fully focuses in the identification and validation of Salmonella serovar specific targets, at the same time graduate students will involve in the identification of different Salmonella serovars from different food sources to be used in the validation of the target primers. Validation involves in-silico across a wide array of Salmonella serovars and V-NTI motif search with in the genome of the same serovars and closely related bacterial species. At the end of this period we will have Salmonella serovars target specific primers ready to be validated in-vitro, for their ultimate utilization of custom microplate array. In the mean time the PI and graduate students functionalize and validate gold and magnetic nanoparticles for the specific capture of target Salmonella serovars from food components.

IMPACT: 2012/09/01 TO 2013/08/31
What was accomplished under these goals? <!-- /* Font Definitions */ @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"?? ??"; mso-font-charset:78; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"?? ??"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-fareast-language:JA;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> The major accomplishments in this one-year period of this grant are mainly preparation of the working conditions for the successful execution of the project objectives. These steps include: 1) Establishment of Food Nanotechnology lab for the effective running of objectives, 2) Collaborative laboratory Visit of the Nano-biosensor laboratory in Michigan State University, and Center for Nano-Biotechnology Research at Alabama State University (ASU); and 3) Retrieval and collection of so far sequenced Salmonella serovars into our genome Database software V-NTI

PUBLICATIONS: 2012/09/01 TO 2013/08/31
No publications reported this period.