IMMUNOMAGNETIC SEPARATION OF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA FROM POTATO PEEL EXTRACTS TO IMPROVE DETECTION SENSITIVITY ON A CRYSTAL VIOLET PECTATE MEDIUM OR BY PCR

Immunomagnetic separation (IMS) procedures for the selective separatio n of Erwinia carotovora subsp. atroseptica from potato peel extract we re optimized for the recovery of target and removal of nontarget bacte ria. A streptomycin-resistant strain of Erw. carotovora subsp. atrosep tica was used in combination with a crystal violet pectate (CVP) mediu m supplemented with 100 mu g ml(-1) of streptomycin to determine the r ecovery level of the target bacterium. Recovery obtained with a polycl onal antiserum against Erw. carotovora subsp, atroseptica at a concent ration of 6 mu g IgG ml(-1) was greater than that obtained with two mo noclonal antibodies against lipopolysaccharides of Erw. carotovora sub sp. atroseptica at a concentration of 10 mu g IgG ml(-1). A linear rel ationship was found between particle concentration ranging from 12 to 200 mu g ml(-1) and recovery level. When the Advanced Magnetics (AM) p rotein A and anti-rabbit IgG particles in the AM separation system and the Dynal anti-rabbit IgG particles in the Dynal separation system we re examined, the highest recovery level per lug of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti-rabbit particles (37%). Without IMS, detection of Erw. caroto vora subsp. atroseptica in tuber peel extracts on a CVP-medium without streptomycin was impossible when the ratio of Erw. carotovora subsp. carotovora to Erw. carotovora subsp. atroseptica was greater than 100 or when large numbers of other saprophytic bacteria were present, beca use of overcrowding. IMS, using the AM anti-rabbit IgG particles, ensu red that Erw. carotovora subsp. atroseptica could be enumerated in tub er peel extract consistently, to a detection level of 100 cells ml(-1) . Similarly, the IMS procedure lowered the detection level of Erw. car otovora subsp. atroseptica in a twofold diluted peel extract by PCR to cn 2.0 x 10(3) cells ml(-1) or 50 cells per reaction tube. In contras t, positive results in PCR without IMS were obtained only when the pee l extract was diluted 100 times and when the concentration of Erw., ca rotovora subsp. atroseptica was at least 10(5) cells ml(-1).