Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (16)
- Analytical Chemistry
- Journal of the American Chemical Society
- Analytical Biochemistry
- Bioinformatics (Oxford, England)
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
- Analytical Chemistry
- Analytical Chemistry
- Analytical Biochemistry
- Journal of Clinical Microbiology
- Analytical Chemistry
- Journal of Clinical Microbiology
- Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology
- Molecular Biotechnology
- Influenza and Other Respiratory Viruses
- Foodborne Pathogens and Disease
Articles by Erica D. Dawson in JoVE
JoVE Immunology and Infection
ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza
InDevR, Inc.
ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.
Other articles by Erica D. Dawson on PubMed
Deuterium Nuclear Magnetic Resonance Spin-lattice Relaxation of Analytically Relevant Solvent Systems
We previously reported that the nuclear magnetic resonance (NMR) 14N spin-lattice relaxation times (T1) of CH3CN in CH3CN-H2O mixtures directly correlate with the solution viscosity when scaled for temperature (eta/T) in this common chromatographic mobile phase system.' Here, we demonstrate that the 2HT1 relaxation times also correlate with viscosity, contrary to a previous report. (2) This establishes 2HT1 relaxation times as a useful means of measuring changes in solution viscosity in CH3CN-H2O mixtures. We show thermal convection to result in grossly decreased, apparent T1's, by as much as approximately 40%, in nonspinning samples. This effect can be eliminated by moderate sample rotation or confinement of the sample to within the rf-irradiated region. The problem of thermal convection is revealed in systems having long Ti's and has implications in T1 experiments employing nonspinning samples at elevated temperatures, including inherently nonspinning systems, such as those used in high-pressure studies.
Probing Transport and Microheterogeneous Solvent Structure in Acetonitrile-water Mixtures and Reversed-phase Chromatographic Media by NMR Quadrupole Relaxation
Mixtures of CH(3)CN and H(2)O are the predominant solvent systems used in reversed-phase liquid chromatographic (RPLC) separations, as well as in a multitude of other applications. In addition, acetonitrile is the simplest model for an amphiphilic molecule possessing both organic and polar functional groups. Although many studies have focused on this solvent system, the general nature of the intermolecular interactions are not fully understood, and a microscopic description of the proposed microheterogeneity that exists is still not clearly established. In the present study, we measure the spin-lattice relaxation times (T(1)) of (14)N to determine reorientational correlation times (tau(c)) of CH(3)CN-H(2)O solvent mixtures over the entire binary composition range and at temperatures ranging from 25.0 to 80.0 degrees C. At all compositions, the microscopic observable, tau(c), is found to be directly proportional to the macroscopic solution viscosity when scaled for temperature (eta/T). This indicates that for a constant composition, this system's dynamics are well described by hydrodynamic theory on a microscopic level. These results suggest that under appropriate conditions, the measurement of changes in quadrupolar relaxation times is a reliable means of determining changes in solution viscosity. We stress the importance of this approach in systems not amenable to traditional viscosity measurements, such as those having species in interfacial regions. This approach is used to examine the changes in the interfacial solution viscosity of CH(3)CN-H(2)O mixtures in contact with a commercially available C(18)-bonded stationary phase. The measurements indicate that CH(3)CN is motionally hindered at the stationary phase surface. The surface affected CH(3)CN has a larger dependence of tau(c) on temperature than the bulk CH(3)CN, indicating greater changes in the interfacial viscosity as a function of temperature. Additionally, the bulk relaxation data show direct correlations to existing models of proposed regions of structure for CH(3)CN-H(2)O mixtures. Using a microscopic hydrodynamic approach, we show that, quite unexpectedly, each of the experimentally determined parameters in the viscosity correlation plots change simultaneously, and we propose that these are indicative of changes in the distribution of species for this microheterogeneous liquid system. Although distinct regions for the onset of microheterogeneity have previously been proposed, within the framework of a microscopic hydrodynamic model and the recently proposed model of Reimers and Hall,(1) the present data support the existence of a microheterogeneous solvent structure that varies continuously over the full range of temperatures and compositions examined.
Spotting Optimization for Oligo Microarrays on Aldehyde-glass
Low-density microarrays that utilize short oligos (<100 nt) for capture are highly attractive for use in diagnostic applications, yet these experiments require strict quality control and meticulous reproducibility. However, a survey of current literature indicates vast inconsistencies in the spotting and processing procedures. In this study, spotting and processing protocols were optimized for aldehyde-functionalized glass substrates. Figures of merit were developed for quantitative comparison of spot quality and reproducibility. Experimental variables examined included oligo concentration in the spotting buffer, composition of the spotting buffer, postspotting "curing" conditions, and postspotting wash conditions. Optimized conditions included the use of 3-4 microM oligo in a 3x standard saline citrate/0.05% sodium dodecyl sulfate/0.001% (3-[(3-cholamidopropyl) dimethylammonia]-1-propane sulfonate) spotting buffer, 24-h postspotting reaction at 100% relative humidity, and a four-step wash procedure. Evaluation of six types of aldehyde-functionalized glass substrates indicated that those manufactured by CEL Associates, Inc. yield the highest oligo coverage.
ConFind: a Robust Tool for Conserved Sequence Identification
SUMMARY: ConFind (conserved region finder) identifies regions of conservation in multiple sequence alignments that can serve as diagnostic targets. Designed to work with a large number of closely related, highly variable sequences, ConFind provides robust handling of alignments containing partial sequences and ambiguous characters. Conserved regions are defined in terms of minimum region length, maximum informational entropy (variability) per position, number of exceptions allowed to the maximum entropy criterion and the minimum number of sequences that must contain a non-ambiguous character at a position to be considered for inclusion in a conserved region. Comparison of the calculated entropy for an alignment of 95 influenza A hemagglutinin sequences with random deletions results in a 98% reduction in the average error in ConFind relative to the 'Find Conserved Regions' option in BioEdit. REQUIREMENTS: ConFind requires Python 2.3, but Python 2.4 or an upgrade of the optparse module to Optik 1.5 is suggested. The program is known to run under Linux and DOS.
Robust Sequence Selection Method Used to Develop the FluChip Diagnostic Microarray for Influenza Virus
DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to identify regions of conservation in the influenza virus genome. From these regions of conservation, capture and label sequences capable of discriminating between different viral types and subtypes were selected. The salient features of the method were the use of phylogenetic trees for data reduction and the selection of a relatively small number of capture and label sequences capable of identifying a broad spectrum of influenza viruses. A detailed experimental evaluation of the selected sequences is described in a companion paper. The software is freely available under the General Public License at http://www.colorado.edu/chemistry/RGHP/software/.
Experimental Evaluation of the FluChip Diagnostic Microarray for Influenza Virus Surveillance
Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed "signatures" for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.
MChip: a Tool for Influenza Surveillance
The design and characterization of a low-density microarray for subtyping influenza A is presented. The microarray consisted of 15 distinct oligonucleotides designed to target only the matrix gene segment of influenza A. An artificial neural network was utilized to automate microarray image interpretation. The neural network was trained to recognize fluorescence image patterns for 68 known influenza viruses and subsequently used to identify 53 unknowns in a blind study that included 39 human patient samples and 14 negative control samples. The assay exhibited a clinical sensitivity of 95% and clinical specificity of 92%.
Identification of A/H5N1 Influenza Viruses Using a Single Gene Diagnostic Microarray
In previous work, a simple diagnostic DNA microarray that targeted only the matrix gene segment of influenza A (MChip) was developed and evaluated with patient samples. In this work, the analytical utility of the MChip for detection and subtyping of an emerging virus was evaluated with a diverse set of A/H5N1 influenza viruses. A total of 43 different highly pathogenic A/H5N1 viral isolates that were collected from diverse geographic locations, including Vietnam, Nigeria, Indonesia, and Kazakhstan, representing human, feline, and a variety of avian infections spanning the time period 2003-2006 were used in this study. A probabilistic artificial neural network was developed for automated microarray image interpretation through pattern recognition. The microarray assay and subsequent subtype assignment by the artificial neural network resulted in correct identification of 24 "unknown" A/H5N1 positive samples with no false positives. Analysis of a data set composed of A/H5N1, A/H3N2, and A/H1N1 positive samples and negative controls resulted in a clinical sensitivity of 97% and a clinical specificity of 100%.
Real-time Quantification of RNA Polymerase Activity Using a "broken Beacon"
A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.
Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza A+B Test for Rapid Diagnosis of Influenza
The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A+B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses.
Diagnostic Microarray for Influenza B Viruses
The importance of global influenza surveillance using simple and rapid diagnostics has been frequently highlighted. For influenza type B, the need exists for discrimination between the two currently circulating major lineages, represented by virus strains B/Victoria/2/87 and B/Yamagata/16/88, as only one of these lineages is represented in seasonal influenza vaccines. Here, the development and characterization of a low-density DNA microarray (designated BChip) designed to detect and identify the two influenza B lineages is presented. The assay involved multiplex nucleic acid amplification and microarray hybridization of viral RNA. Detection and lineage identification was achieved in less than 8 h. In a study of 62 influenza B virus samples from 19 countries, dating from 1945 to 2005, as well as 5 negative control samples, the assay exhibited 97% sensitivity and 100% specificity. Furthermore, application of a trained artificial neural network to the pattern of relative fluorescence signals resulted in correct lineage assignment for 94% of 50 applicable influenza B viruses, with no false assignments.
Evaluation of MChip with Historic Subtype H1N1 Influenza A Viruses, Including the 1918 "Spanish Flu" Strain
The robustness of a recently developed diagnostic microarray for influenza, the MChip, was evaluated with 16 historic subtype H1N1 influenza A viruses (A/H1N1), including A/Brevig Mission/1/1918. The matrix gene segments from all 16 viruses were successfully detected on the array. An artificial neural network trained with temporally related A/H1N1 viruses identified A/Brevig Mission/1/1918 as influenza virus A/H1N1 with 94% probability.
Detection of Adamantane-resistant Influenza on a Microarray
Influenza A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza therapeutics, thereby complicating treatment decisions.
Molecular Detection of Streptococcus Pyogenes and Streptococcus Dysgalactiae Subsp. Equisimilis
We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2-3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.
MChip, a Low Density Microarray, Differentiates Among Seasonal Human H1N1, North American Swine H1N1, and the 2009 Pandemic H1N1
The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in <8 hours for under US$10.
Identification of Escherichia Coli O157 by Using a Novel Colorimetric Detection Method with DNA Microarrays
Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.
