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High Throughput, Rapid, Foodborne Pathogen Screening

Bhunia, Arun K
Purdue University
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The overarching objective is to develop and optimize biosensor- based assays for sensitive detection of multiple foodborne pathogens from food. The specific aims are:

AIM 1. To optimize and improve sample preparation step prior to biosensor-based detection

AIM 2. Improve biosensor performance by employing novel probes, reagents and media

AIM 3. Optimize biosensor performance for pathogens

More information

Foodborne diseases are responsible for approximately 48 million illnesses with 3000 deaths annually in the US (Scallan et al., 2011). There are over 200 known microbial, chemical or physical agents that can result in illness when consumed (Newell et al., 2010). Of these, microbial source comprising of bacterial, viral and fungal are of major concern. CDC estimates that of all the foodborne infections, 44% of the hospitalizations and deaths are attributed to 31 known pathogens (Scallan et al., 2011). Among these pathogens, the majority of the illnesses, hospitalizations and deaths are caused by five known pathogens, which include Norovirus, nontyphoidal Salmonella, Clostridium perfringens, Campylobacter spp. and Staphylococcus aureus. Salmonella enterica causes gastroenteritis in humans and nearly 1 million people in the US are infected annually, resulting in approximately 19,000 hospitalizations and over 378 deaths (Scallan et al., 2011).

For biosensor technologies to work effectively with varieties of food matrices, sample preparation strategy and sample enrichment steps must be well defined. For culturing multiple pathogens in the same media, we have developed a multipathogen enrichment broth (SEL; Salmonella, E. coli, Listeria) that supports growth of these three pathogens simultaneously. We will determine various antibodies and mammalian cell receptors that are used by pathogens during infection as potential ligands for pathogen capture on biosensor platforms. As we have demonstrated earlier, mammalian Hsp60 protein, a receptor for L. monocytogenes LAP (Listeria adhesion protein) worked very well on a fiber optic sensor (Koo et al., 2011) and on a microfluidic biochip platform (Koo et al., 2009) for direct detection of this pathogen. Application of other pathogen-specific receptor molecules including translocated intimin receptor (TIR) for capture of Shiga-toxin producing E. coli (STEC) strains, and ?1-Integrin for capture of Yersinia, Shigella and Salmonella, etc. will be studied in this project. For cloning of TIR, the gene will be PCR amplified from E. coli EDL933 (O157:H7) and inserted into pET-SUMO vector (Invitrogen) for protein expression (Jagadeesan et al., 2011). His-tagged proteins will be purified using Ni-affinity column. The optical fibers coated with antibody or receptor is first exposed to sample containing target pathogens for capture. Subsequently, pathogen-specific second antibody/aptamer/receptor conjugated to a fluorophor (Cy5, Alexa Fluor) will be added for pathogen-specific signal. The formation of pathogen-antibody/receptor sandwich will emit fluorescence that generates evanescent wave, which is detected by a built-in laser detector (Analyte 2000, Research International, Monroe, WA). Previously, we have developed fiber optic-based assays for each L. monocytogenes (Geng et al., 2004; Mendonca et al., 2012; Nanduri et al., 2006; Ohk et al., 2010), E. coli O157:H7 (Geng et al., 2006) and Salmonella enterica serovar Enteritidis (Valadez et al., 2009) and validated the assays with food products.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Bacterial Pathogens
Natural Toxins