Automated immunomagnetic separation and microarray detection of E. coli O157:H7 from poultry carcass rinse.
We describe the development and application of an electromagnetic flow cell and fluidics system for automated immunomagnetic separation (IMS) of Escherichia coli O157:H7 directly from poultry carcass rinse. We further describe the biochemical coupling of automated sample preparation with nucleic acid microarrays. Both the cell concentration system and microarray detection method did not require cell growth or enrichment from the poultry carcass rinse prior to IMS. Highly porous Ni foam was used to enhance the magnetic field gradient within the flow path, providing a mechanism for immobilizing immunomagnetic particles throughout the fluid rather than the tubing wall. A maximum of 32% recovery efficiency of non-pathogenic E. coli was achieved within the automated system with 6 s cell contact times using commercially available antibodies targeted against the O and K antigens. A 15-min protocol (from sample injection though elution) provided a cell recovery efficiency that was statistically similar to > I h batch captures. O157:H7 cells were reproducibly isolated directly from poultry carcass rinse with 39% recovery efficiency at 10(3) CFU ml(-1) inoculum. Direct plating of washed beads showed positive recovery of O157:H7 directly from poultry carcass rinse at an inoculum of 10 CFU ml(-1). Recovered beads were used for direct polymerase chain reaction (PCR) amplification and microarray detection, with a process-level detection limit (automated cell concentration though microarray detection) of < 10(3)CFU ml(-1) in poultry carcass rinse.
Chandler
D P
DP
Analytical Microbiology Group, Pacific Northwest National Laboratory, Richland, WA 99352, USA. dp.chandler@pnl.gov
Brown
J
J
Call
D R
DR
Wunschel
S
S
Grate
J W
JW
Holman
D A
DA
Olson
L
L
Stottlemyre
M S
MS
Bruckner-Lea
C J
CJ
D000818:Animals; D001431:Bacteriological Techniques; D002645:Chickens; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity