Submitted to: United States-Japan Cooperative Program in Natural Resources (UJNR)
Publication Type: Proceedings
Publication Acceptance Date: 8/27/2017
Publication Date: 11/12/2017
Citation: Gehring, A.G., Armstrong, C.M., Capobianco Jr, J.A., He, Y., Paoli, G., Fratamico, P.M. 2017. Rapid and potentially portable detection and quantification technologies for foodborne pathogens . United States-Japan Cooperative Program in Natural Resources (UJNR). p.12.
Technical Abstract: Introduction Traditional microbial culture methods are able to detect and identify a single specific bacterium, but may require days or weeks and typically do not produce quantitative data. The quest for faster, quantitative results has spurred development of “rapid methods” which usually employ biorecognition elements and computer interfaced instrumentation for microbial detection and exhibit assay times from minutes to hours . Generally, a biosensor, a device or process which produces, amplifies, and measures a signal when the target species binds to a biorecognition element (often a biological molecule such as an antibody or nucleic acid), exhibits rapid response, high specificity, general immunity from interference, and low detection limits . However, the high levels of non-target microorganisms and other materials in food samples do sometimes interfere with biosensor operation, and the detection limits required for food safety (e.g., a single cell in 325 g of food for “zero tolerance” microorganisms such as E. coli O157:H7 or Listeria monocytogenes) are even lower than can be achieved with most biosensors. Our five year research project plan focuses on the development of inexpensive, simplistic, small footprint, and robust biosensor-based methods that may have practical application in the field. Our objectives address all of the above concerns to implement such a portable device that effectively prepares and presents concentrated target pathogens for specific and sensitive detection and/or identification near-line or inline during food production. This “preview” will cover the three key objectives that we propose to address in our five year research project plan. They include 1) sample preparation with emphasis on the non-growth enrichment, quantitative concentration of bacterial cells in food samples, 2) development and validation of biosensor-based rapid methods for the detection of specific bacterial targets, and 3) development and validation of rapid identification methods for the typing and confirmation of bacterial targets. The ultimate goal is to implement all of these techniques in the field in as short of a time, preferably = 8 h or a single work shift. Physical separation and concentration of foodborne bacteria The desired detection limit for many pathogens in food is orders of magnitude lower than the detection limit (103-106 CFU/mL) of available rapid methods. This disparity between the desired and available detection limits is currently addressed by the use of culture enrichment to raise the concentration of target pathogen(s) above the assay detection limit. However, enrichment adds hours or days to the assay (delaying remediation or recall), is problematical when multiple pathogens must be detected, and prevents quantitative determination of the original pathogen concentration. These and other problems are well known, and physical separation methods such as immunoaffinity capture, flow-through centrifugation and filtration are frequently identified as potential alternatives to enrichment . Practical physical separation methods must process 100-1000 mL of food homogenate and concentrate a significant proportion of the target bacteria into a volume of 10-100 uL at reasonable cost in less than an hour. When coupled to a rapid multiplexed detection system such as real-time PCR, such methods could enable quantitative detection of multiple pathogens in a single food sample at 1 CFU/g within two hours of sampling. This would represent an order of magnitude reduction in response time relative to enrichment-based assays, and allow the full potential of modern rapid methods to be realized. A widely-used separation approach is immuno-adsorption on antibody-coated paramagnetic particles or “immunomagnetic beads” (IMBs). While rapid, simple, readily automated, and capable of selectively concentrating the target