Biomedical subjects

Carolyn E Meador

Publications and source records attributed to Carolyn E Meador.

3 recordsLinked to original sources

PubMed2006

Fluorescent signal amplification of carbocyanine dyes using engineered viral nanoparticles.

We report enhancement in the fluorescent signal of the carbocyanine dye Cy5 by using an engineered virus as a scaffold to attach >40 Cy5 reporter molecules at fixed locations on the viral capsid. Although cyanine dye loading is often accompanied by fluorescence quenching, our results demonstrate that organized spatial distribution of Cy5 reporter molecules on the capsid obviates this commonly encountered problem. In addition, we observe energy transfer from the virus to adducted dye molecules, resulting in a highly fluorescent viral nanoparticle. We have used this enhanced fluorescence for the detection of DNA-DNA hybridization. When compared with the most often used detection methods in a microarray-based genotyping assay for Vibrio cholerae O139, these viral nanoparticles markedly increased assay sensitivity, thus demonstrating their applicability for existing DNA microarray protocols.

Avidin↗

PubMed2005

Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp.

The morbidity and mortality associated with Vibrio-mediated waterborne diseases necessitates the development of sensitive detection technologies that are able to elucidate the identity, potential pathogenicity, susceptibility, and viability of contaminating bacteria in a timely manner. For this purpose, we have designed a single multiplex PCR assay to simultaneously amplify 95 diagnostic regions (encompassing species/serogroup-specific, antimicrobial resistance, and known toxin markers) and combined it with a long oligonucleotide microarray to create a platform capable of rapidly detecting and discriminating the major human pathogenic species from the genus Vibrio: V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus. We were able to validate this strategy by testing 100 geographically and temporally distributed isolates and observed an excellent concordance between species- and serotype-level microarray-based identification and traditional typing methods. In addition to accurate identification, the microarray simultaneously provided evidence of antibiotic resistance genes and mobile genetic elements, such as sulfamethoxazole-trimethoprim constins and class I integrons, and common toxin (ctxAB, rtxA, hap, hlyA, tl, tdh, trh, vvhA, vlly, and vmhA) and pathogenicity (tcpA, type III secretion system) genes that are associated with pathogenic Vibrio. The versatility of this method was further underscored by its ability to detect the expression of known toxin and virulence genes from potentially harmful viable but nonculturable organisms. The results suggest that this molecular identification method provides rapid and definitive information that would be of value in epidemiological, environmental, and health risk assessment surveillance.

Anti-Infective Agents↗

PubMed2004

Nucleic acid amplification strategies for DNA microarray-based pathogen detection.

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Antigens, Bacterial↗