title: Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry.
abstract: A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.
authors.affiliation: U S Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079, USA. dan.buzatu@fda.hhs.gov
authors.forename: Dan A
authors.initials: DA
authors.lastname: Buzatu
authors.forename: Willie M
authors.initials: WM
authors.lastname: Cooper
authors.forename: Christine
authors.initials: C
authors.lastname: Summage-West
authors.forename: John B
authors.initials: JB
authors.lastname: Sutherland
authors.forename: Anna J
authors.initials: AJ
authors.lastname: Williams
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Bass
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Smith
authors.forename: Robert S
authors.initials: RS
authors.lastname: Woodruff
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Christman
authors.forename: Steven
authors.initials: S
authors.lastname: Reid
authors.forename: Randal K
authors.initials: RK
authors.lastname: Tucker
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Haney
authors.forename: Ashfaqe
authors.initials: A
authors.lastname: Ahmed
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Rafii
authors.forename: Jon G
authors.initials: JG
authors.lastname: Wilkes
mesh_terms: D003257:Consumer Product Safety; D010697:Eosine I Bluish / Q000494:pharmacology*; D019453:Escherichia coli O157 / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification* / Q000528:radiation effects; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D038761:Photobleaching; D017319:Photosensitizing Agents / Q000494:pharmacology*; D018724:Spinacia oleracea / Q000382:microbiology*
chemical_list: D017319:Photosensitizing Agents; D010697:Eosine I Bluish
keywords: Escherichia coli O157:H7; Flow cytometry; Food matrix interference; Photobleaching; RAPID-B; Same day analysis