Carbon Nanoparticles as Detection Labels in Antibody Microarrays. Detection of Genes Encoding Virulence Factors in Shiga Toxin-Producing Escherichia coli

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2...

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Bibliographic Details
Published in:	Analytical chemistry (Washington) Vol. 83; no. 22; pp. 8531 - 8536
Main Authors:	Noguera, Patricia S, Posthuma-Trumpie, Geertruida A, van Tuil, Marc, van der Wal, Fimme J, Boer, Albert de, Moers, Antoine P. H. A, van Amerongen, Aart
Format:	Journal Article
Language:	English
Published:	Washington, DC American Chemical Society 15-11-2011
Subjects:	agreement Analytical chemistry assay bacteria Carbon - chemistry Chemistry dna microarray E coli Exact sciences and technology General, instrumentation Genes Immunoassay Immunoassay - methods Miscellaneous Nanoparticles Nanoparticles - chemistry Oligonucleotide Array Sequence Analysis - methods optimization pathogen detection Polymerase Chain Reaction quantification Shiga Toxin - biosynthesis Shiga-Toxigenic Escherichia coli - genetics Shiga-Toxigenic Escherichia coli - metabolism Shiga-Toxigenic Escherichia coli - pathogenicity Staining and Labeling - methods statistics tests Toxins Virulence Factors - genetics Performance evaluation Nanoparticle Escherichia coli Virulence Microanalysis Optimization Immunological method Design Specificity Gene Efficiency Detection limit Bacteria Genetics Feces Ungulata Nucleic acid Enterobacteriaceae Bovine Antibody Use Chemical sensor Carbon Polymerase chain reaction Screening Toxin Vertebrata Sensitivity Mammalia DNA Artiodactyla Detection Sensor array
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Summary:	The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box–Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-2700 1520-6882
DOI:	10.1021/ac201823v