Source: HJ Science & Technology, Inc. submitted to NRP
PORTABLE AUTOMATION TECHNOLOGY FOR RAPID DETECTION OF FOODBORNE PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1009468
Grant No.
2016-33610-25359
Cumulative Award Amt.
$99,997.00
Proposal No.
2016-01046
Multistate No.
(N/A)
Project Start Date
Jul 15, 2016
Project End Date
Mar 14, 2018
Grant Year
2016
Program Code
[8.5]- Food Science & Nutrition
Recipient Organization
HJ Science & Technology, Inc.
187 Saratoga Avenue
Santa Clara,CA 95050
Performing Department
(N/A)
Non Technical Summary
According to a Centers for Disease Control and Prevention (CDC) report in 2011, approximately 48 million Americans get sick, 128,000 are hospitalized and 3,000 die each year from food poisoning, also known as foodborne illness. Bacteria are the source of many food poisoning cases because they can multiply and spread in foods that are contaminated. Symptoms of food poisoning can vary and develop as quickly as thirty minutes to up to several days after eating the infected food. Rapid methods for detection of these foodborne pathogens are necessary to protect the public health and to ensure food safety. Current detection methods rely on having to take the samples to the laboratory for analysis, which is often a time consuming, costly and laborious process. More importantly, the lack of real-time data hampers proper and timely decision making. The proposed portable detection instrument with automation is capable of rapid detections of foodborne pathogens such as E. coli O157:H7 and salmonella with selectivity and sensitivity that can only be achieved today with laboratory-based manually performed procedures. As such, the proposed technology will help ensure a safe food supply by improving our ability to detect foodborne pathogens, and reduce the incidence of foodborne illnesses and death. To this end, the proposed research effort will satisfy one of the five primary USDA NIFA Societal Challenge Areas: Food Safety.
Animal Health Component
75%
Research Effort Categories
Basic
0%
Applied
75%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7237410202050%
7125010104050%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
7410 - General technology; 5010 - Food;

Field Of Science
1040 - Molecular biology; 2020 - Engineering;
Goals / Objectives
The overall goal of this SBIR research project is to demonstrate the feasibility of a novel portable microfluidic automation technology capable of rapid detection of foodborne pathogens during pre- and post-harvest processing and distribution. Specifically, we apply our patent-pending microfluidic automation technology as a platform to two well established laboratory-based pathogen separation and detection methods: immunomagnetic separation (IMS) and real time polymerase chain reaction (qPCR). By integrating IMS and qPCR and including automated sample loading into a microfluidic format, we expect to detect as few as 1 cfu/25g of food with an enrichment time of 4 hours and a combined enrichment and analysis end-to-end time of 5 hours. In addition, we expect to perform rapid foodborne pathogen detections with sensitivity and specificity that are currently only achievable with laboratory-based manually performed procedures.
Project Methods
The proposed technology is the result of applying our patent-pending microfluidic automation technology as a platform to integrate immunomagnetic separation (IMS) and real time polymerase chain reaction (qPCR). To achieve the technical objectives of the proposed microfluidic automation technology for rapid detection of foodborne pathogens, the Phase I technical work plan includes 1) develop and calibrate microfluidic real-time PCR assay for E. coli O157, 2) Perform integrated IMS and real-time PCR, 3) perform integrated IMS and real-time PCR using beef samples spiked with E. coli O157, 4) perform integrated IMS and real-time PCR using beef samples spiked with E. coli O157 with enrichment, 5) automate sample loading, 6) improve capture probe affinity using aptamers. During the subsequent Phase II effort, we will expand and scale the Phase I design to include 1) detection capabilities for additional foodborne pathogens, and 2) multi-sample detection capability.

Progress 07/15/16 to 03/14/18

Outputs
Target Audience:Dr. Erik Jensen,the project PI,met with researchers at the USDA Western Regional Research Center in Albany, CAto discuss the objectives and results of our Phase I effort. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In our successful SBIR Phase I effort, we have established the technical feasibility of our patented valveless fluidic switching (VLFS) microfluidic automation technology to perform rapid detection of 1 colony forming unit (cfu) of pathogenic E. coli O157:H7 in 25g food samples with an overall sample processing and analysis time of 6 hours. Specifically, we have adapted a commercial immunomagnetic separation (IMS) kit (Dynabeads anti-E. coli O157, Thermofisher Scientific) to our portable microfluidic automation platform and developed a miniaturized qPCR system for isolation and detection of E. coli O157. Both the microfluidic IMS and qPCR have been demonstrated to have equivalent performance to their respective manually performed benchtop procedures. As such, we have achieved the overall Phase I goal of demonstrating microfluidic automation of rapid foodborne pathogen detection by accomplishing each of the following four technical objectives: 1) We have demonstrated rapid detection of E. coli O157:H7 in phosphate buffered saline (PBS) using our VLFS based microfluidic IMS and qPCR platform with comparable results to manually performed benchtop methods. The manual and automated IMS procedures resulted in comparable % recoveries (23% and 32%, respectively), which are both similar to those found in literature. In addition to the automated cell purification procedure, we developed a method for automated thermal lysis of cells during the IMS procedure to isolate genetic material from the magnetic beads which are inhibitory to the microfluidic qPCR. To compare our microfluidic qPCR with the performance of a MX3005p commercial qPCR platform, we tested samples ranging between 1000 and 10 copies of E. coli O157 genomic DNA and negative controls. Our microfluidic qPCR has identical performance for limit of detection and improved performance for amplification efficiency. 2) We have demonstrated rapid detection of E. coli O157 in real food samples by using beef spiked with E. coli O157:H7 and automated sample loading of enrichment media. Specifically, we are able to detect 10 E. coli O157 cells spiked onto 25 g beef samples with a 5 hour 10 minute overall time to result, corresponding to a ~6 hour time to result for 1 cfu/25g sample. Our novel, automated loading technology enables direct transfer of enrichment broth to the pathogen detection platform without any pipetting steps. 3) We have evaluated the use of aptamers as capturing agents for possible improvements in assay performance compared to antibody based capturing agents. However, the results suggest that antibodies have better assay performances than aptamers. 4) Based on the results of Phase I, we have designed a Phase II microfluidic device and prototype instrument capable of automating each of the following steps: Sample loading, IMS, elution/cell lysis, mastermix preparation, and qPCR analysis. In addition, the Phase II prototype is designed to perform analysis of three enriched samples simultaneously, and is capable of detecting multiple foodborne pathogens. As such, the completion of the above objectives demonstrates the technical feasibility of rapid, automated, portable foodborne pathogen detection using our VLFS and miniaturized qPCR technology with sensitivity and specificity only currently achievable with laboratory-based instruments.

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