Develop a DNA microarray-based assay for analysis for the most common microbial foodborne pathogens in the U.S., their virulence factors and antibiotic resistance genes. <P> Our main aim is to unify identification and characterization of the major microbial foodborne pathogens in one DNA microarray chip (DNA chip). The target foodborne pathogens of this project are: Staphylococcus aureus, Bacillus cereus, Listeria, Coliforms, Salmonella, Shigella, E. coli, Clostridium botulinum, Clostridium perfringens, Yersinia, Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus.
NON-TECHNICAL SUMMARY: Foodborne pathogens are a major public health concern and cause large economic losses for U.S. agriculture. Rapid detection and identification of such pathogens may allow corrective measures to be taken early on, thereby reducing foodborne illness and economic losses. In recent years, microarray technology (DNA chip) has developed rapidly. These chips can contain many thousands of different gene sequences representing many different pathogens, so they can be used for identify any one of many pathogens. The overall goal of this project is to develop a DNA chip-based assay to identify which pathogen is contaminating the food, and to determine which virulence factors the pathogen contains and which antibiotics it is resistant to. Our aim is to unify identification and characterization of the fourteen major microbial foodborne pathogens in one DNA microarray chip. This will simplify testing of contaminated foods. Our approach is to design short DNA molecules (20-30 bases) corresponding to the genes that are unique to each pathogen, genes that determine which toxins or virulence factors a pathogen can produce, and genes that determine which antibiotics a pathogen is resistant to. These short molecules are attached to a glass slide (chip) in specific positions. When the sample of contaminated food is analyzed, the genes of the pathogen will bind to the correspobnding genes located at fixed positions on the chip. This will allow the tester to identify and characterize the foodborne pathogen.
APPROACH: Our approach is based on designing short oligonucleotide probes (20-30 bases) corresponding to unique rDNA, virulence factor and antibiotic resistance gene sequences, to immobilize them on the glass slides, each in a specific position, creating a DNA chip. We propose to use multi-target PCR amplification for amplifying and labeling many target sequences simultaneously either by applying universal PCR primers, multiplex, DOP PCR or uniplex PCR. The amplified-labeled DNA then will be hybridized to the DNA chip surface. Each amplified sequence will anneal to its corresponding oligonucleotide on the chip. The pathogen can then be identified based on the position on the chip where the PCR-labeled target DNA annealed. This new DNA chip may enable analysis of multiple foodborne pathogens and their virulence factors simultaneously.
PROGRESS: 2001/09 TO 2004/08<BR>
The overall aim of the project was to develop a food safety DNA microarray for analysis of food microbial pathogens including Staphylococcus aureus, Bacillus spp., Listeria, Campylobacter, Clostridium perfringens, Salmonella, Shigella and E. coli. The project had four objectives: 1. Developing species-specific microarrays for identification of foodborne bacterial pathogens 2. Developing PCR amplification and fluorescent labeling of bacterial pathogens for microarray analysis including a safety control system 3. Integration of the species-specific microarrays into a multipathogen DNA chip 4. Development of a microarray for analysis of microbial antibiotic resistance 5. These objectives were largely achieved, as described below and in nine published manuscripts. 1. We developed microarrays for analysis of Listeria , Campylobacter , Clostridium perfringens , Salmonella, Shigella and E. coli , Staphylococcal enterotoxins  and Bacillus spp.. These methods were tested with numerous microbial strains and were able to provide accurate identification of these pathogens including identification of their major virulence factors. 2. Several methods of PCR amplification and fluorescent labeling of microbial pathogens for microarray analysis were developed including universal primers, asymmetric PCR, single strand generation and chemical labeling. These methods are described in the above publications. 3. We integrated these arrays into a single DNA chip . This multipathogen oligonucleotide microarray was used for simultaneous analysis of S. aureus enterotoxin genes, Listeria spp., Campylobacter spp. and Clostridium perfringens toxin genes using a single chip. 4. We also developed a microarray for analysis for erythromycin resistance  which was tested with many S. aureus strains. 5. Our work required extensive DNA sequence analysis and oligoprobe design, but there were no widely available programs suitable for our purposes. To improve oligoprobe selection and design we developed a computer program . The program selects the best set of primers and oligonucleotide probes (based on the user specification) by using data from multiple gene alignments. The program also carries out a tiling analysis routine to design probes for detection of single base mutations. The program significantly simplifies and automates the development of microarrays for microbial diagnostics. Because of emerging public health concerns and FDA needs, we expanded our research to include analysis of Bacillus anthracis , which was not part of our original proposal. Our current work continues to focus on the important Bacillus cereus group of pathogens, as an additional component of our existing microarray for analysis of Salmonella, Shigella and E. coli . Thus in this project we developed a food safety microarray for bacterial food pathogens, technology to amplify and label microbial DNA, and computational tools for microarray development.
IMPACT: 2001/09 TO 2004/08<BR>
The DNA microarrays for analysis of food microbial pathogens we developed can be used for detection o the most common food microbial pathogens