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Enrichment and detection of Escherichia coli O157:H7 from water samples using an antibody modified microfluidic chip.

ABSTRACT: Low abundant (<100 cells mL(-1)) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices, each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using benchtop real-time quantitative polymerase chain reaction (PCR), targeting genes which effectively discriminated the O157:H7 serotype from other nonpathogenic bacteria. The recovery of target cells from water samples was determined to be approximately 72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and wastewater samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies.

SUBMITTER: Dharmasiri U

PROVIDER: S-EPMC2859668 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Enrichment and detection of Escherichia coli O157:H7 from water samples using an antibody modified microfluidic chip.

Dharmasiri Udara U Witek Małgorzata A MA Adams Andre A AA Osiri John K JK Hupert Mateusz L ML Bianchi Thomas S TS Roelke Daniel L DL Soper Steven A SA

Analytical chemistry 20100401 7

Low abundant (<100 cells mL(-1)) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices, each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel ...[more]

PMID: 20218574

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Project description:Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment. To achieve this detection, a multipathogen selective enrichment medium is essential to allow the concurrent growth of pathogens. SEL broth was formulated to allow the simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The results were compared to those obtained with the respective individual selective enrichment broths, Rappaport-Vassiliadis (RV) for S. enterica, modified E. coli broth with 20 mg of novobiocin/liter for E. coli O157:H7, and Fraser broth for L. monocytogenes, and a currently used universal preenrichment broth (UPB). The growth of each pathogen in SEL inoculated at 10(1) or 10(3) CFU/ml was superior to that in the respective individual enrichment broth, except in the case of RV, in which Salmonella cells inoculated at both concentrations grew equally well. In mixed-culture experiments with cells of the three species present in equal concentrations or at a 1:10:1,000 ratio, the overall growth was proportional to the initial inoculation levels; however, the growth of L. monocytogenes was markedly suppressed when cells of this species were present at lower concentrations than those of the other two species. Further, SEL was able to resuscitate acid- and cold-stressed cells, and recovery was comparable to that in nonselective tryptic soy broth containing 6% yeast extract but superior to that in the respective individual selective broths. SEL promoted the growth of all three pathogens in a mixture in ready-to-eat salami and in turkey meat samples. Moreover, each pathogen was readily detected by a pathogen-specific immunochromatographic lateral-flow or multiplex PCR assay. Even though the growth of each pathogen in SEL was comparable to that in UPB, SEL inhibited greater numbers of nontarget organisms than did UPB. In summary, SEL was demonstrated to be a promising new multiplex selective enrichment broth for the detection of the three most prominent food-borne pathogens by antibody- or nucleic acid-based methods.

Dataset Information

Enrichment and detection of Escherichia coli O157:H7 from water samples using an antibody modified microfluidic chip.

Publications

Enrichment and detection of Escherichia coli O157:H7 from water samples using an antibody modified microfluidic chip.

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