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Simultaneous detection of multiple food safety threat agents using multiplex PCR and PCR-based microarray approaches

Award No.: 
Principal Investigator: 
Woubit Salah Abdela
PI Organization: 
Tuskegee University
Food-borne pathogens cause millions of clinical illnesses every year and cost billions of dollars to manage and control. Several recent examples including Salmonella enterica serovar Saintpaul in produce, Escherichia coli O157:H7 in ground beef, Listeria monocytogenes in ready to eat meat have led elected officials and consumer groups to call on the food industry and regulatory agencies to prevent contaminated foods from reaching the consumer (Kennedy, 2008). Intentional contamination of food in the form of a biological attack is an even more alarming prospect. This could involve contamination of water. Product tampering or contaminating food supplies is an ever-present danger. Unfortunately, no amount of testing can ensure that every food item is free from accidental or intentional contamination. Conventional culture methods for detection of pathogens are time consuming and results are frequently not available until the food has been either released to the market or consumed. One of the principal tools emerging for the rapid detection of pathogens is the polymerase chain reaction (PCR). Developing rapid PCR methods to substitute for the culture method is a pressing issue in clinical and food hygiene tests. Routine multiplex PCR techniques are limited by the amplifiable PCR product sizes and the maximum number of pathogens that could be detected simultaneously. This limited throughput does not allow the identification of agents and their closely related variants for the purpose of classifying and tracing origin of contamination. In this proposal, our goal is to establish, modify, optimize and validate multiplex and quantitative Real Time-PCR methods to detect food safety threats. Specifically, we aim to establish the proof-of-principle that in the future will allow us to expand the system to more agents and to test the system in real world samples. We have designed preliminary 96-well PCR-microarray platforms based on specific DNA sequences to enable simultaneous detections of the agents. Each of the 96-wells will represent a specific agent of food safety threat, and PCR data from each well will be quantitatively recorded in real time. PCR-based high throughput approach is a novel and unique aspect of our concept that once successfully established it can be used either as a primary or confirmatory diagnostic test at a larger scale. By achieving these aims, we will establish a multiplex-detection method that can be further developed to improve the sensitivity, specificity and throughput capacity of the detection of multiple high-impact food-borne pathogens simultaneously.
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