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Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

ABSTRACT: The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment.

SUBMITTER: Zimmer-Faust AG

PROVIDER: S-EPMC3993297 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

Zimmer-Faust Amity G AG Thulsiraj Vanessa V Ferguson Donna D Jay Jennifer A JA

Applied and environmental microbiology 20140221 9

The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the rel ...[more]

PMID: 24561583

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Project description:A versatile method is presented to form dendrimer superstructures by exploiting coacervate-core micelles as a template to confine and organize the hyperbranched macromolecules. First, complex coacervate-core micelles are formed from negative-neutral block copolymers and positively charged polyamidoamine dendrimers. The dendrimers inside the micellar core are then covalently cross-linked with each other upon addition of glutaraldehyde. After removal of the block copolymer from the assembly by increasing the salt concentration, consecutively, the formed Schiff bases cross-linking the dendrimers are reduced to amines, followed by a final dialysis step. This leads to well-defined covalently cross-linked nanostructures, coined dendroids, with a size of around 30 nm in diameter and a molecular weight of approximately 2.5 MDa. By incorporating dendrimer-encapsulated gold nanoparticles (AuDENs) into the micelle template strategy, the aggregation number of dendrimers inside the dendroids is determined by counting the nanoparticles in TEM micrographs. Furthermore, TEM performed at different tilt angles and AFM analysis corroborate formation of stable, covalently linked three-dimensional structures. Reconstruction of the TEM tilt series results in a tomogram further illustrating the 3D distribution of the gold nanoparticles, and hence the individual dendrimers, in the nanostructure. These dendroids appear to have a hard, poorly compressible core and a relatively soft outside. The versatility of the hierarchical building up of the supermolecules is illustrated by the controlled and synchronous incorporation of empty dendrimers and AuDENs into a single hybrid dendroid structure. The presented strategy allows for the preparation of a variety of classes of supermolecules, depending on the type of micellar-core macromolecule, e.g., dendrimer, cross-linker, and nanoparticles, used. Considering the broad interest in dendrimers as well as micelles in a plethora of research areas, e.g., (targeted) drug delivery, biomedical imaging, theragnostics, and catalysis, there is a great potential for dendroids and related classes of covalently linked macromolecules, viz., supermolecules.

Dataset Information

Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

Publications

Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

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