ampliphox : Technology Introduction

Transcription

1 ampliphox : Technology Introduction Rapid, Inexpensive Colorimetric Detection Technology for Low-Density Microarrays Erica Dawson, PhD VP Research and Development dawson@indevr.com

2 Portfolio of Innovative Technologies Rapid Virus Quantification ViroCyt 2100 Virus Counter Chip-Based Disease Research ampliphox colorimetric detection technology for low density microarrays based on photopolymerization Variety of early stage projects focused on selfcontained, automated diagnostic platforms PLIX automated cartridge-based platform for low density microarray diagnostics Low-cost liquid handling robot and assay cartridge for genotyping of influenza viruses Early stage, game-changing technology for protein quantification 2

3 ampliphox Colorimetric Detection Platform consists of small instrument (5 cube) and reagent kit End result is an array of macroscopic spots visible to the eye Time to result for detection process of ~20 min Microarray detection of any biotinylated target Analytical sensitivity equivalent to Cy3 fluorescence Cost is ~10x lower than typical fluorescence scanner Final result 3

4 Photopolymerization 4

5 ampliphox Software Automated Image Analysis User creates a layout mask Automated pattern recognition based on signal and bkgd characteristics for each spot Logic defined by the user automates identification for user-defined patterns

6 Custom Microarrays New Product Offering Nucleic acid and protein/antibody array printing service Array placement compatible with ampliphox (other custom formats available upon request) Min. spot diameter of 100 µm; Max. ~1700 spots/array Pricing based on number of arrays/slide and number of unique captures/slide Quick turnaround time for custom quote generation

7 Contact Us For More Information Website: Telephone:(303) InDevR, Inc Central Ave. Suite 106 Boulder, CO USA

8 Using ampliphox and low-density DNA microarrays to genotype the foodborne pathogen Shiga toxigenic Escherichia coli Beatriz Quiñones, Ph.D. Research Molecular Biologist U.S. Department of Agriculture Agricultural Research Service Western Regional Research Center Produce Safety and Microbiology Research Unit Albany, California

9 DNA Microarrays for Pathogen Detection DNA Microarrays: - Sequence-specific probes are uniformly attached to a glass surface 1. Sample preparation Bacterial Cell Isolation Nucleic acids purification 2. Microarray fabrication Probe choice Probe functionalization Amplification (optional) Printing/spotting Labeling Blocking 3. Hybridization y1 y4 y3 y2 y5 x1 x2 x3 x4 x5 4. Scanning 5. Data processing Quantification Analysis Biological meaning

10 Microarray Detection Methods Fluorescent Assays: Enzymatic incorporation of fluorescent dye during DNA amplification Direct labeling with Cy3/Cy5 nucleotides Indirect labeling with aminoallyl-dutp and fluorescent dye Inconsistent incorporation of dye-label Expensive non-portable microarray scanners Colorimetric Assays: Use a streptavidin-conjugated substrate with biotin-labeled target on the microarray Silver staining Alkaline phosphatase Unstable reagents requiring controlled temperatures Variable development time leading to overexposure

11 Shiga Toxin-Producing Escherichia coli (STEC) O157:H7 E. coli O157:H7 recent outbreaks linked to foodborne & waterborne sources Ground beef was associated with most outbreaks Produce-associated outbreaks may be due to higher consumption of fruits and vegetables From 1995 to 2011, 25 outbreaks in the United States were associated with lettuce and other leafy vegetables More than 50% of produce-associated outbreaks traced to leafy vegetables grown on the central coast of California

12 Gene Targets for O157 STEC Detection stx1 and stx2: Shiga toxins 1 and 2 - Major virulence factors in E. coli O157 pathogenicity - AB 5 cytotoxins linked to hemolytic-uremic syndrome eae: intimin adherence outer membrane protein - Adhesin for binding to infected human cells - eae + strains are associated with severe human illness per: perosamine synthetase - Enzyme required for the O157-antigen synthesis

13 Microarray Detection of O157 STEC with ampliphox Ec RM1697 stx1 + /stx2 + eae + /per + Ec RM1600 stx1 + /stx2 - eae + /per + Ec RM6011 stx1 - /stx2 + eae + /per + Ec RM4876 stx1 - /stx2 - eae + /per + Array Layout control stx1 stx2 per eae Ec RM1273 stx1 - /stx2 - eae + /per + Ec RM3654 stx1 - /stx2 - eae + /per - Ec RM3655 stx1 - /stx2 - eae - /per - Ec K-12 stx1 - /stx2 - eae - / per - Negative control The patterns of photopolymerization correlated with the strain genotype (Quiñones, B., et al., 2011, Foodborne Pathog. Dis., 8: )

14 Average SNR Values Quantification of O157 STEC Detection Specificity stx1 stx2 per eae 20 0 Positive signals had average signal-to-noise ratio (SNR) values above 50 while negative signals had SNR values less than 2.5 (Quiñones, B., et al., 2011, Foodborne Pathog. Dis., 8: )

15 Average SNR values Quantification of O157 STEC Detection Sensitivity stx1 stx2 per eae CFU / ml of E. coli O157:H7 strain RM1697 The detection sensitivity for using photopolymerization is CFU/ml (Quiñones, B., et al., 2011, Foodborne Pathog. Dis., 8: )

16 Percentage of Isolates Selection of Target Probes for Non O157 STEC Detection Human Isolates of STEC Non O157 by Serogroup, United States N=940 isolates 55 O-groups, each 1% % other ND other und O Group (Brooks, et al., 2005, J. Infect. Dis., 192: ) Certain non O157 STEC strains are potentially as virulent as O157 strains

17 Additional Virulence Probes for STEC Detection 1. Effectors secreted by the type III secretion system (nlea and ent/espl2) 2. Other virulence factors saa: STEC autoagglutinating adhesin suba: subtilase AB 5 cytotoxin ehxa: enterohemolysin espp: extracellular serine protease katp: catalase peroxidase

18 DNA Microarray-Based STEC Detection with ampliphox Colorimetric Method O26 O26 O111 O111 O104 O104 gadb gadb Group 1 O45 O45 O121 O121 O91 O91 O128 O128 Group 2 O103 O103 O145 O145 O113 O113 O157 O157 Group 3 ent ent nlea nlea espp espp saa saa katp katp suba suba eae eae per per ehxa ehxa stx1 stx1 flic H7 flic H7 stx2 stx2 Group 4 O Groups (11) O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, O157 E. coli control (1) gadb Virulence Factors (10) eae, ehxa, ent/espl2, espp, katp, nlea, saa, suba, stx1, stx2 ampliphox control (Quiñones, B., et al., 2012, Front. Cell. Inf. Microbio. 2:61)

19 Detection of O157 and non-o157 STEC with ampliphox and DNA Microarrays (Quiñones, B., et al., 2012, Front. Cell. Inf. Microbio. 2:61)

20 Sampling Regions for STEC Salinas Valley area considered the salad bowl of the United States major produce production region USDA/WRRC Monterey San Benito Sampling done in 3 counties in California: Monterrey, San Luis Obispo, and San Benito San Luis Obispo Sample locations in regions for leafy vegetable production Fields (23) Ranches (12) Other locations (47): adjacent waterways and habitats

21 (Quiñones, B., et al., 2012, Front. Cell. Inf. Microbio. 2:61)

22 Conclusions ampliphox colorimetric technology is a rapid and sensitive colorimetric method for STEC detection on DNA microarrays. Validation tests showed photopolymerization was highly specific and had a sensitivity of CFU/ml. In addition to O157 STEC detection, our DNA microarray method accurately genotyped STEC non O157 isolates from produce production regions in California.

23 Acknowledgments USDA Western Regional Research Center Produce Safety & Microbiology Unit Albany, California Michelle Swimley Koh-Eun Narm Ronak Patel Michael Cooley Robert Mandrell InDevR, Inc., Boulder, Colorado Erica Dawson Amber Taylor University of California, Davis Edward Atwill Michelle Jay-Russell