Rapid and sensitive isolation of Campylobacter jejuni using immunomagnetic separation from patient specimens exposed to oxygen

This study describes a method for detecting Campylobacter jejuni in patient stools with subsequent isolation using antibody-magnetic beads in conjunction with selective culture and PCR. Monoclonal antibodies specific for the flagellin A and major outer membrane protein of C. jejuni were generated; two clones (1C7 and 4B2) were used to coat magnetic beads for immunomagnetic separation (IMS). C. jejuni strain NCTC11168 was recovered from human stool samples spiked with varying concentrations (10(1)-10(5) CFU/mL) by Campylobacter (Campy)-IMS or a conventional culture-based method and plated on modified charcoal-cefoperazone-deoxycholate agar; the number of colonies was enumerated. The detection limits of Campy-IMS and conventional culture-based method with spiked stool samples were 10(2) and 10(4) CFU/mL, respectively. The sensitivity of IMS-PCR was 10-10,000-fold higher than that of direct PCR. The recovery rate of C. jejuni from spiked stools stored for 12 to 72 h decreased from 72.3 to 5.9% with Campy-IMS and from 48.5 to 0.1% with the conventional culture-based method. Importantly, of 20 PCR (+)/bacterial culture (-) samples that were diagnosed as probable cases according to general criteria, 95% (19/20) were confirmed positive by Campy-IMS. Thus, this study suggests a solution to overcome the problems caused by the inconsistency between probable and confirmed cases of Campylobacter infection.