AFFINITY CONCENTRATION OF LISTERIA MONOCYTOGENES CELLS ON CONCANAVALIN A‐COATED POLYESTER CLOTH AND SUBSEQUENT DETECTION BY THE POLYMERASE CHAIN REACTION

Abstract

The Canavalia ensiformis lectin concanavalin A (con A) was immobilized on macroporous polyester cloth to form an inexpensive high surface area adsorbent (con A‐cloth) for the affinity concentration of bacterial cells in aqueous suspensions. When the con A‐cloth was packed into a 1 ml pipette tip, the resulting mini‐column could be used to concentrate large volumes of dilute L. monocytogenes cell suspensions, followed by the polymerase chain reaction (PCR) amplification of L. monocytogenes‐specific hly A sequences from lysates of the captured cells. This improved the effective sensitivity of the PCR as compared to the assay of L. monocytogenes in unconcentrated suspensions. Several enrichment broths (Fraser Broth, Listeria Enrichment Broth and Brain Heart Infusion Broth) were found to be inhibitory to the PCR of L. monocytogenes when introduced directly into the reaction mixture. This inhibitory effect was completely eliminated when the L. monocytogenes cells were captured on the con A‐cloth and washed to remove the enrichment broth components prior to performing the PCR. Since the lectin con A is reactive with a broad variety of Gram positive and Gram negative bacteria, this simple and inexpensive affinity concentration method should be applicable to the PCR detection of other pathogens in enrichment cultures.