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 separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medium supplemented with 100 micrograms ml-1 of streptomycin to determine the recovery level of the target bacterium. Recovery obtained with a polyclonal antiserum against Erw. carotovora subsp. atroseptica at a concentration of 6 micrograms IgG ml-1 was greater than that obtained with two monoclonal antibodies against lipopolysaccharides of Erw. carotovora subsp. atroseptica at a concentration of 10 micrograms IgG ml-1. A linear relationship was found between particle concentration ranging from 12 to 200 micrograms ml-1 and recovery level. When the Advanced Magnetics (AM) protein A and anti-rabbit IgG particles in the AM separation system and the Dynal anti-rabbit IgG particles in the Dynal separation system were examined, the highest recovery level per microgram of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti-rabbit particles (37%). Without IMS, detection of Erw. carotovora 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, because of overcrowding. IMS, using the AM anti-rabbit IgG particles, ensured that Erw. carotovora subsp. atroseptica could be enumerated in tuber peel extract consistently, to a detection level of 100 cells ml-1. Similarly, the IMS procedure lowered the detection level of Erw. carotovora subsp. atroseptica in a twofold diluted peel extract by PCR to ca 2.0 x 10(3) cells ml-1 or 50 cells per reaction tube. In contrast, positive results in PCR without IMS were obtained only when the peel extract was diluted 100 times and when the concentration of Erw. carotovora subsp. atroseptica was at least 10(5) cell ml-1.
van der Wolf
J M
JM
DLO Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.
Hyman
L J
LJ
Jones
D A
DA
Grevesse
C
C
van Beckhoven
J R
JR
van Vuurde
J W
JW
Pérombelon
M C
MC
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005069:Evaluation Studies as Topic; D018189:Immunomagnetic Separation; D008070:Lipopolysaccharides / Q000276:immunology; D008969:Molecular Sequence Data; D016973:Pectobacterium carotovorum / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000382:microbiology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004269:DNA, Bacterial; D008070:Lipopolysaccharides