Testing and validation of high density resequencing microarray for broad range biothreat agents detection.

Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assay's limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 10(4) copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally available from multiplexed RT-PCR/PCR-based detection assays.

Universal detection and identification of avian influenza virus by use of resequencing microarrays.

Zoonotic microbes have historically been, and continue to emerge as, threats to human health. The recent outbreaks of highly pathogenic avian influenza virus in bird populations and the appearance of some human infections have increased the concern of a possible new influenza pandemic, which highlights the need for broad-spectrum detection methods for rapidly identifying the spread or outbreak of all variants of avian influenza virus. In this study, we demonstrate that high-density resequencing pathogen microarrays (RPM) can be such a tool. The results from 37 influenza virus isolates show that the RPM platform is an effective means for detecting and subtyping influenza virus, while simultaneously providing sequence information for strain resolution, pathogenicity, and drug resistance without additional analysis. This study establishes that the RPM platform is a broad-spectrum pathogen detection and surveillance tool for monitoring the circulation of prevalent influenza viruses in the poultry industry and in wild birds or incidental exposures and infections in humans.

Comparison of detection and signal amplification methods for DNA microarrays.

One of the factors limiting the use of DNA microarray technology for the detection of pathogenic organisms from clinical and environmental matrices has been inadequate assay sensitivity. To assess the effectiveness of post-hybridization secondary detection steps to enhance the sensitivity of DNA microarray-based pathogen detection, we evaluated a panel of 11 commercial and novel hybridization detection and signal amplification methods (direct labeling, indirect aminoallyl labeling, antibody, DNA dendrimers, viral particles, internally fluorescent nanoparticles, tyramide signal amplification, resonance light scattering nanoparticles and quantum dots) using a multiplex PCR and spotted long oligonucleotide microarray for Vibrio cholerae. Quantitative parameters such as sensitivity, signal intensity, background, assay complexity, time and cost were assessed and provide comparative criteria to be considered for DNA microarray experimental design. While the most important parameter is likely to vary based on the assay, when weighted equally, the findings suggest that recognition element- and dye-functionalized viral particles provide the most attractive option for microarray detection and signal amplification.

Virulence gene- and pandemic group-specific marker profiling of clinical Vibrio parahaemolyticus isolates.

Vibrio parahaemolyticus is a halophilic bacterium capable of causing food- and waterborne gastroenteritis, wound infections, and septicemia in humans. The organism has recently received increasing attention, as the emergence of a new clone, V. parahaemolyticus O3:K6, has resulted in the first documented pandemic spread of V. parahaemolyticus. We used microarray analyses to explore the presence of known virulence factors and genetic markers thought to be specific for V. parahaemolyticus O3:K6 and its clonal derivatives. Analyses of 48 human clinical isolates collected between 1997 and 2005 revealed that the V. parahaemolyticus chromosome 2 type III secretion system is not specifically associated with pandemic strains and can be found in tdh-negative (i.e., Kanagawa-negative) clinical isolates. These results highlight the genetic dynamism of V. parahaemolyticus and aid in refining the genetic definition of the pandemic group members.

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.

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.

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.