T1-12 Microplate Immunocapture (IMC): A New Solution for the Isolation/Concentration of Escherichia coli O157:H7 from Food Samples

Monday, August 4, 2014: 11:45 AM
Room 111-112 (Indiana Convention Center)
Patrice Arbault, BioAdvantage Consulting, Orlienas, France
Delphine Larose, Nexidia, Dijon, France
Nicolas Desroche, Nexidia, Dijon, France
Jean Guzzo, Nexidia, Dijon, France
Introduction: Immunocapture (IMC) is used for the concentration of pathogenic bacteria from food product to facilitate their detection. Immuno-magnetic separation has been traditionally the reference method for capturing foodborne pathogen bacteria.

Purpose: We have developed a new method for the immunocapture of pathogenic bacteria based on 96-well microplate coated with specific antibodies. The objective of this study was to demonstrate the efficiency of this method in capturing Escherichia coli O157 in enriched food samples.

Methods: After adding 100µl of enrichment broth (BPW) into the wells, antibodies are capturing present target bacteria. Then, after a washing step, a subculture is performed (3 to 5 hours) in the wells with addition of fresh enrichment medium (100µl mTSBn) for increasing the number of target bacteria. The efficiency of the system was assessed by performing immunocapture on food samples (ground beef / raw milk cheese, 25-g sample size), artificially contaminated with E. coli O157:H7. Naturally contaminated beef samples found positive by PCR screening were also confirmed by IMC plate. Tests were also done with magnetic beads.

Results: For ground beef samples, IMC with microplate allowed the capture of about 5.4 105 CFU of E. coli O157 when magnetic beads recovered 2.7 103 CFU. For both raw milk cheeses, IMC microplate captured about 3.2 105 CFU (goat cheese) and 2.6 105 CFU (Mont d’Or) of E. coli O157 when magnetic beads recovered about 6.2 103 CFU (goat cheese) and 2.7.104CFU (Mont d’Or). IMC microplate allowed the confirmation of 33 out of 38 positive PCR screening for the naturally contaminated meat samples.

Significance: IMC microplate was shown effective for the capture isolation of E. coli O157 bacteria from enriched meat samples. The subculture step done into the wells, after immunocapture, contributes to a good confirmation rate.