SBIR-STTR Award

Most contemporary diagnostic tests are designed to detect and identify a single particular pathogen if it may be present in a given specimen. Furthermore such assays typically rely upon a short biomarker, or short signature gene sequence element to INDIRECTLY determine if the specimen is or is not present in the specimen. Such assays are inevitably vulnerable to false negative or false positive reports that can have costly implications from poorly informed decisions in critical food safety situations. In distinct contrast the DIRECT determination of multiple gene sequences from one or more target pathogens can provide unequivocal evidence for the presence or absence of multiple pathogens that may be associated with a particular food safety-related specimen. Furthermore such gene sequences, as opposed to a measured signal intensity from a traditional biomarker assay, provide direct information on the specific strains and variants of pathogens that may be detected, and identification in such detail to support forensic epidemiology or tracking of a chain of breakdowns food-safety. The re-sequencing microarray is a DIRECT DNA sequencing technology that is most efficient at providing accurate gene sequences for hundreds of target pathogen genes in each assay. This Phase I project will develop a prototype resequencing microarray-based diagnostic assay for general application in food-safety. If superior assay performance is demonstrated and validated as may be expected, then this prototype assay has excellent potential for commercialization in broad food-safety related applications. The product will demonstrate significantly superior performance compared to traditional microbial culture or more recent molecular diagnostic assays (e.g. PCR). OBJECTIVES: We will design and validate a 47 kb microarray-based resequencing assay to detect and identify approximately 20 high priority, viral and bacterial foodborne disease agents. Norovirus is highest priority viral pathogen of the assay. Bacterial genera that are targets of the assay include Brucella, Campylobacter, Clostridium, Enterococcus, Escherichia, Listeria, Mycobacterium, Salmonells, Shigella, Vibrio and Yersinia. Two to three loci (segments of targeted pathogen gene sequences) will be detected per agent, and 500-1,000 bases of targeted pathogen gene sequence will be interrogated per locus. The resequencing microarray will represent such gene sequences representing selected prototype strains and variants of targeted pathogens. However, the assay results will distinguish and specify differences of gene sequences of pathogens detected in the specimen from these prototype pathogen gene sequences that are programmed onto the microarrays. Synthetic templates that are perfectly complementary (or by design partially complementary) to the sequences on the array design will be utilized for validation testing of the new microarray based assay. Assay performance metrics including sensitivity, specificity, false positive rate and false negative rates will be determined. Therefore, the ultimate goal of Phase I is to demonstrate that the foodborne pathogen diagnostic assay correctly detects, identifies and differentiaties synthetic DNA templates representing targeted pathogens in laboratory samples. Technical feasibility will be demonstrated by the following key milestones: 1) Resequencing microarrays designed by TessArae scientists and manufactured by Affymetrix. Microarrays will pass standard Affymetrix quality assurance metrics. (Month 3) 2) Laboratory validation completed using synthetic templates. Assay metrics including sensitivity (<10e4 genomic equivalents) and specificity (>95%) are demonstrated. Zero false positive rate is demonstrated. (Month 8). APPROACH: The agents to be detected by the assay include species specific high priority areas identified by USDA. In addition, the assay will be designed to detect a number of other foodborne viral and bacterial pathogens. Detection is based upon the capability of the assay to utilize genomic material of one or more targeted pathogens in a specimen as template for microarray resequencing of target genes. Thus the first stage of assay design, after selection of target pathogens, is to identify pathogen specific gene sequences suitable for identification and differentiation of strains and variants of the target pathogens from unrelated species, or other microbes that may be unrelated to food safety issues but genetically similar to the targeted pathogens. When multiple strains of a targeted pathogen are at issue, prototype gene sequences of particular strains may be selected to be similar to sequences of the plurality of known variants. This bioinformatics-intensive effort relies upon combined search of public genome databases and recent literature related to food safety microbiology. Once an aggregate of 47,974 bp of targeted pathogen gene sequences are determined, the information is forwarded to the microarray manufacturer for production of the first lot of microarrays. Meanwhile, each targeted pathogen gene sequence to be represented on the pending microarrays will be analyzed to select suitable flanking oligonucleotide primer sequences for amplification. Candidate amplification primers will be validated first in singlet reactions, by direct detection of specific amplification products from the synthetic oligonucleotide templates. Then, multiplex mixtures of amplification products will be evaluated to confirm amplification of individual components in simple or complex mixtures of the synthetic templates. Once validated, multiplex amplification reactions will be used with mixtures of single or multiple synthetic templates at different specified concentrations to establish detection sensitivity. Parallel experiments with deliberately matching or mismatching templates will be used to establish assay specificity. Finally a pilot study is undertaken using a series of deliberately blinded preparations of synthetic templates representing different target pathogens at varied concentrations. Once this level of validation is achieved during the Phase I SBIR effort, the assay is ready for limited production and distribution for applications of food safety monitoring, screening, surveillance and outbreak epidemiology - anticipated substance of a follow-on Phase II SBIR proposal

The primary objectives of this Phase II project are to 1) validate performance of the prototype resequencing pathogen microarray application for detection and identification of selected food-borne pathogens, including varieties of viruses, bacteria and eukaryotic agents; 2) iterate and port the prototype application to a more practical, "market-friendly" product form factor that requires less expensive capital equipment to use, and that anticipates significantly lower operating cost per assay, without compromise of assay multiplicity, sensitivity or specificity; and 3) perform limited additional performance validation of the product application to enable initial commercial marketing to government and private sector, domestic and international laboratories that provide food-safety testing services. OBJECTIVES: The purpose of this proposal is to validate a new diagnostic approach for detection and strain-level identification of multiple microorganisms that may be associated with any particular outbreak of food-related illness. Preliminary results from the Phase I prototype and characterization demonstrate that the microarray-based diagnostic platform is capable of detecting multiple agents, both viral and bacterial, simultaneously and definitively, with a zero false positive rate. We have conducted pilot experiments in collaboration with the FDA Center for Food Safety and Applied Nutrition (CFSAN) using archived specimens from "real-world" samples. The results showed that the assay was able to identify pathotype/serotype within a species and perform strain-level discrimination within a pathotype. The proposed validation studies are a natural extension of this clear proof-of-concept demonstration and will involve three specific aims: 1) Conduct performance analysis and validation of the prototype RPM-FS1.0 food-borne pathogen diagnostic assay, using "real world" samples; 2) Iterate the initial design of the R&D prototype RPM-FS1.0 and port the platform to a more end user-friendly and marketable RPM-FS2.0 configuration; and 3) Conduct pre-market validation of the RPM-FS2.0 product. Validation studies will be performed at TessArae and CFSAN under an existing Research Collaboration Agreement. Upon completion of the proposed RPM-FS2.0 validation studies, the Phase II proposal supports beta-testing of the food-borne pathogen diagnostic assay by interested government and private sector customers. By the conclusion of the Phase II project, most of the necessary evaluations and validation work will be completed to support certification of the microarray-based food-borne screening and diagnostic platform for use in government and commercial food-borne pathogen testing laboratories. APPROACH: Specific Aim 1: Conduct performance analysis and validation of the prototype RPM-FS1.0 food-borne pathogen diagnostic assay using "real world" samples. The prototype application uses multiple pathogen genes per category of targeted pathogens. The pathogen targets of the prototype RPM-FS1.0 application are: 1) Viruses, including Adenovirus (subgroup F), Astrovirus (8 serotypes), Calicivirus (noro- and sapoviruses, multiple clades), Hepatitis A and E viruses (multiple types, clades) and Rotaviruses (types A, B, C); 2) Bacteria, including Bacillus (B. cereus, B. licheniformis and B. subtilis), Brucella, Campylobacter, Citrobacter, Clostridium (C. botulinum, C. difficile, C. perfringens), Cronobacter sakazakii, Enterococcus (E. faecalis, E. faecium), Escherichia coli (including multiple enteropathogenic toxin genes), Klebsiella (K. oxytoca, K. pneumoniae), Listeria monocytogenes, Mycobacterium paratuberculosis, Plesiomonas shigelloides, Providencia, Pseudomonas aeruginosa, Salmonella enteritidis (including multiple serovar determinants), Shigella, Staphylococcus aureus (including multiple toxin genes), Streptococcus (S. pyogenes, S zooepidemicus), Vibrio (V. cholerae, V. parahaemolyticus, V. vulnificus), Yersinia (Y. enterocolitica, Y. pseudotuberculosis); and 3) Eukaryotic pathogens, including Giardia, Cryptosporidium Entamoeba, Cyclospora, Toxoplasma. Analytical sensitivity validation, as a determination of the assay's limit of detection (LoD) for a target pathogen, typically involves a large number of replicate assays across a range of serial dilutions from titrated stocks of positive control pathogens. We will leverage the multiplex economy of the RPM platform by execution of LoD determinations with selected mixtures and dilutions of multiple RPM-FS1.0 target pathogens. Specific Aim 2: Iterate the initial design of the prototype RPM-FS1.0 (single unit 169- array/wafer cartridge microarray) and port the platform to a "market-friendly" RPM-FS2.0 configuration (289-array/wafer, pegged microarray strip format). The recently released Affymetrix GeneAtlas instrument and pegged array system offers significant reductions of end-user entry costs, individual assay costs (microarray and reagents), and "hands-on" labor in assay execution. The iterated product RPM-FS2.0 assay design will retain target pathogen/target gene assay components of RPM-FS1.0 that are judged in prior studies to meet performance benchmarks. We do anticipate that implementation of the new product configuration will require refinement of the sample-processing protocol (wetware) using the GeneAtlas hybridization-wash-stain station. Specific Aim 3: Conduct pre-market validation of the iterated RPM-FS2.0 design. In general, we project that validation of the product RPM-FS2.0 will require similar scope and scale of assays as described above for the work plan for Specific Aim 1. However, we do not anticipate that complete repetition of these validation assays will be required for those target pathogen gene detector tiles that are ported unchanged to the newproduct array configuration