Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry |
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Authors: | Dan A Buzatu Willie M Cooper Christine Summage-West John B Sutherland Anna J Williams Deborah A Bass Lisa L Smith Robert S Woodruff Jessica M Christman Steven Reid Randal K Tucker Christopher J Haney Ashfaqe Ahmed Fatemeh Rafii Jon G Wilkes |
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Affiliation: | 1. U. S. Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079, USA;2. U. S. Food and Drug Administration, Arkansas Regional Laboratory, Jefferson, AR 72079, USA;3. U. S. Food and Drug Administration, Center for Tobacco Products, Rockville, MD 20850, USA |
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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. |
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Keywords: | Flow cytometry RAPID-B Food matrix interference Photobleaching Same day analysis Escherichia coli O157:H7 |
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