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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED BIOSENSOR-BASED PROCESSES FOR MULTIPATHOGENIC ANALYTE DETECTION
2010 Annual Report


1a.Objectives (from AD-416)
The ultimate goal of this project is to develop rapid, specific, and sensitive biosensor-based assays for diverse pathogenic bacteria which can be widely adopted in applications ranging from simple field tests to high speed, high throughput laboratory screening assays. To meet this goal, several objectives will be pursued: 1) Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. 2) Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. 3) Develop integrated assay systems based on multiple target biosensor platforms.


1b.Approach (from AD-416)
The primary objective of the proposed research is to develop biosensor processes that are capable of detecting multiple pathogens of food safety and food security concern. We plan to concentrate our research on a few selected pathogens: E. coli O157:H7, Listeria monocytogenes, Salmonella and Yersinia spp. Methods will generally be developed with culture media as the sample matrix, and then extended to food samples containing the target pathogen. The efficacy of developed methods will be primarily tested in ground beef, ready-to-eat meats and liquid eggs. Modification of the plan to include other pathogens and foods will be determined by ARS needs. To facilitate the progress of planned research, we will seek useful advice and/or input from our colleagues in other Research Units at the Eastern Regional Research Center (ERRC).


3.Progress Report
Traditional methods for the detection of foodborne pathogens involve the growth of microorganisms in selective culture media (enrichment) followed by microbiological and biochemical characterization of putative pathogenic isolates which can take days to weeks to complete. Furthermore, these methods are designed to detection a single pathogen. This project is aimed at developing rapid methods for the detection of the very low numbers of pathogens in food and the development of methods to detect multiple pathogens simultaneously. To reduce the dependence on culture enrichment and decrease the time-to-result, methods for concentration of foodborne pathogens were developed. A novel and rapid filtration method was developed for the concentration of E. coli O157:H7 from extracts of ground beef. When coupled with real-time PCR the method was very sensitive and rapid (detection of 2 cells per gram in less than 3 hours). Another pathogen concentration method employed immunomagnetic beads (IMBs, microscopic magnetic beads with attached antibodies that bind to specific pathogens) to capture pathogens from food. A mixed antibody IMB was generated that allow the capture of both E. coli O157:H7 and Salmonella, which, when coupled with fluorescence assay, allowed the rapid and sensitive detection of the two pathogens from foods. Additional progress was made on an antibody-based method with the potential for the detection of numerous pathogens and associated toxins (antibody microarray). In this method capture antibodies are spotted on a surface and the captured pathogens or toxins are detected using a second, fluorescently labeled antibody. Through a series of empirical investigations, the microarray assay detection limit was improved 100-fold to ~10,000 cells per milliliter, the total assay time was decreased 5-fold to ~30 min, and the assay cost was greatly reduced. DNA-based methods were also developed for the detection and differentiation of multiple pathogens. For effective and reliable detection of foodborne pathogens, a multiplex real-time PCR combined with multi-pathogen culture enrichment strategy was developed to simultaneously detect E. coli O157, Salmonella, and Listeria monocytogenes. The method was successfully applied to the detection of these three pathogens in a variety of artificially and naturally contaminated meats. Another multiplex real-time PCR assay was developed for the simultaneous detection and differentiation of the 3 pathogenic species of Yersinia. When the method was applied to the detection of Yersinia species from foods artificially contaminated with each or all of the 3 species of Yersinia, 2 of the species were detected with high sensitivity. The third species, which grows much slower than the others, was detected with 10- to 100-fold less sensitivity. Another contribution was the development of a real-time PCR protocol and algorithm for the facile estimation of DNA concentration of samples with varying PCR efficiencies. This method, which uses as little as one concentration of a DNA standard and three dilutions of the unknown DNA sample, will be important when accurate enumeration is desired.


4.Accomplishments
1. Developed process capable of simultaneous detection of E. coli O157:H7 and Salmonella. According to the Centers for Disease Control and Prevention, each year foodborne pathogens result in 76 million cases of illness, more than 300,000 hospitalizations and 5,000 deaths. Current microbiological methods for pathogen detection are laborious and time consuming. Thus, there is a need to develop rapid, effective and accurate alternative approaches to detect the presence of pathogens in foods. To meet this goal, ARS scientists in Wyndmoor, PA, have developed an approach involving the use of immunomagnetic beads (IMBs, microscopic magnetic particles coated with antibody molecules that bind to specific pathogens) for pathogen capture followed by sensitive detection using a method called time-resolved fluorescence (TRF). In current study, a new approach was devised to equip the IMB with two different antibodies and thus capacity to capture both E. coli O157:H7 and Salmonella. Applications of this new IMB allowed the capture and detection of the two pathogens simultaneously from ground beef and spinach samples. This cost-saving and effective pathogen capture and detection process will be of value to regulatory agencies and food testing laboratories.

2. Developed antibody-based microarray method for simultaneous detection of multiple foodborne pathogens. Since traditional microbiological methods are slow (time frame in days), rapid methods (minutes to hours) are needed for the detection of harmful bacteria (pathogens) in foods. Furthermore, it is particularly advantageous if the method can detect multiple pathogens as well as be applied in a high-throughput (simultaneously testing of multiple samples) manner. Antibody microarrays, a technique that uses biological recognition molecules (i.e., antibodies that specifically bind to bacteria or toxins) printed in 100s to 1000s of tiny, arrayed dots, can be used for multiplexed, rapid screening of pathogens. In this method, the array of antibodies that are spotted on a surface capture pathogens or toxins which are detected using a second, fluorescently labeled antibody. ARS scientists in Wyndmoor, PA, developed antibody microarrays for the simultaneous detection of E. coli O157:H7, Salmonella, and E. coli Shiga-like toxin, the antibody microarrays were constructed in 96-well plates, allowing the high throughput processing of up to 96 samples per plate. Upon optimization, the method was able to detect as little as 10,000 cells per milliliter in a total assay time of about 30 min. The assay cost was greatly reduced through the use of relatively inexpensive polystyrene plastic 96-well plates and elimination of the need for chemical modification (biotinylation) of antibodies prior to spotting. This method, which can further multiplexed for the detection of additional pathogens, may be applied by regulatory agencies and food producers for the screening of foods for harmful contaminants.

3. New and versatile DNA-based method for simultaneous detection of multiple foodborne pathogens. Most commonly employed methods for detection of foodborne pathogens only allow the detection of one pathogen at a time. ARS scientists in Wyndmoor, PA, developed, optimized, and evaluated multiplex real-time PCR assay for the simultaneous detection of three major foodborne pathogens, E. coli O157, Salmonella, and Listeria monocytogenes. The PCR assay also included an internal control that indicated the success of the reaction, even in the absence of the pathogens, reducing the likelihood of false-negative results. A universal culture enrichment strategy was used to grow all three pathogens to detectable levels from the very low numbers that might be present in foods. The reliable and sensitive culture enrichment and multiplex real-time PCR assay was successfully applied to various meat products for effective screening for single or multiple pathogens. The ability to detect multiple pathogens in a single assay will save time and resources, increasing our capacity to ensure food safety and public health.

4. Three hour assay for E. coli O157:H7 in ground beef. Rapid methods for the detection of E. coli O157:H7 are desired by food producers and regulatory agencies. Virtually all methods for detection of pathogens in foods currently require culture enrichment of the food sample prior to the detection step, significantly increasing the time-to-result. Approaches that avoid enrichment could provide quantitative, unbiased, and more rapid detection of multiple pathogens. Concentration of the bacteria from food rinses via filtration is an attractive alternative to culture enrichment. A large volume filtration methodology was developed and optimized by ARS scientists in Wyndmoor, PA, that resulted in rapid concentration of E. coli O157:H7 from ground beef samples. A rapid DNA extraction method was also developed that provided high recovery of DNA in a volume of 10 microliters. Coupling the filtration and DNA extraction methods with real-time PCR (polymerase chain reaction) resulted in quantitative detection of E. coli O157:H7 in ground beef at the level of 2 cells per gram of food in less than 3 hours. Further development is expected to yield 30 minute sample preparation and PCR detection within 45 minutes for detection of E. coli O157:H7 and simultaneous detection of 3 or 4 pathogens in a wide variety of foods in less than 2 hours. The developed method significantly decreases the time-to-result in comparison to methods currently employed by food testing laboratories.


Review Publications
Ricardi, J., Haavig, D., Cruz, L., Paoli, G., Gehring, A.G. 2010. Evaluation of the MIT RMID 1000 system for the identification of Listeria species:AOAC performance tested method 090325. Journal of Association of Official Analytical Chemists International. 93(1):249-258.

Suo, B., He, Y., Tu, S., Shi, X. 2010. A multiplex real-time PCR for simultaneous detection of Salmonella spp., E. coli O157 and L. monocytogenes in meat products. Foodborne Pathogens and Disease. 7(6):619-628.

Chen, J., Zhang, L., Paoli, G., Tu, S., Shi, X. 2010. A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis. International Journal of Food Microbiology. 137:168-174.

Suo, B., He, Y., Paoli, G., Gehring, A.G., Tu, S., Shi, X. 2009. Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens. Molecular and Cellular Probes. 24:77-86.

He, Y., Yao, X., Gunther, N.W., Tu, S., Shi, X. 2010. Simultaneous detection and differentiation of Campylobacter jejuni, C. coli, and C. lari in chickens by multiplex real-time PCR. Journal of Food Analytical Methods. 137:168-174.

Last Modified: 10/23/2014
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