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United States Department of Agriculture

Agricultural Research Service

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

Location: Molecular Characterization of Foodborne Pathogens

Title: A coupled anti-L. monocytgenes scFv immunomagnetic bead and PCR method for capture and detection of L. monocytogenes from food

Authors
item PAOLI, GEORGE
item TU, SHU-I
item Fan, Yiling - SHANGHAI JIAOTONG UNIV.
item Shi, Xianming - SHANGHAI JIAOTONG UNIV.

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 7, 2007
Publication Date: May 14, 2008
Citation: Paoli, G., Tu, S., Fan, Y., Shi, X. 2008. A coupled anti-L. monocytgenes scFv immunomagnetic bead and PCR method for capture and detection of L. monocytogenes from food . Meeting Abstract.

Technical Abstract: Listeria monocytogenes is a deadly food-borne pathogen. As the bacterium is quite sensitive to heat, L. monocytogenes is of concern primarily in ready-to-eat foods that are not cooked prior to consumption. As a result, U.S. regulatory agencies have established a "zero-tolerance" policy for L. monocytogenes in ready-to-eat foods as well as policies aimed at reducing or eliminating L. monocytogenes from food processing facilities. Implementation of these policies requires sensitive, rapid and specific tests for the bacterium, driving development of alternatives to slow culture methods. Specificity is critical because only L. monocytogenes is pathogenic to humans and the five other species of Listeria are widely distributed in the environment. Rapid biosensor methods are available for a number of food-borne pathogens, but, in the case of L. monocytogenes, their application has been hampered by the lack of polyclonal serum or monoclonal antibodies specific for L. monocytogenes at the species level. We have isolated an L. monocytogenes-specific single-chain antibody (scFv) from an antibody phage display library. A plasmid vector was constructed that allowed the expression of biotinylated scFvs in Escherichia coli and purification by nickel affinity chromatorgraphy. Anti-L. monocytogenes immunomagnetic beads (IMBs) were generated using the biotinylated scFvs and streptavidin-magnetic beads. The anti-L. monocytogenes scFv-IMBs demonstrated higher efficiencies and improved specificity of capture for L. monocytogenes than were observed for commercial anti-Listeria IMBs. The capture of L. monocytogenes by the anti-Listeria monoctyogenes IMBs is now being coupled with a PCR assay for the specific and rapid detection of L. monocytogenes from food.

Last Modified: 9/10/2014