Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method.
The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.
DeCory
Thomas R
TR
Department of Food Science and Technology, Cornell University, Geneva.
Durst
Richard A
RA
Zimmerman
Scott J
SJ
Garringer
Linda A
LA
Paluca
Gary
G
DeCory
Heleen H
HH
Montagna
Richard A
RA
D001628:Beverages / Q000382:microbiology*; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005374:Filtration / Q000379:methods; D005453:Fluorescence; D005618:Fresh Water / Q000382:microbiology*; D007118:Immunoassay / Q000379:methods*; D018189:Immunomagnetic Separation / Q000379:methods*; D008081:Liposomes; D027845:Malus / Q000382:microbiology*; D008851:Micropore Filters; D012235:Rhodamines; D013997:Time Factors
D008081:Liposomes; D012235:Rhodamines; C022027:lissamine rhodamine B