Kulesh, David A.
Most widely held works by David A Kulesh
Identification of Genomic Signatures for the Design of Assays for the Detection and Monitoring of Anthrax Threats ( Book )
2 editions published between 2004 and 2005 in English and held by 1 library worldwide
Sequences that are present in a given species or strain while absent from or different in any other organisms can be used to distinguish the target organism from other related or un-related species. Such DNA signatures are particularly important for the identification of genetic source of drug resistance of a strain or for the detection of organisms that can be used as biological agents in warfare or terrorism. Most approaches used to find DNA signatures are laboratory based, require a great deal of effort and can only distinguish between two organisms at a time. We propose a more efficient and cost-effective bioinformatics approach that allows identification of genomic fingerprints for a target organism. We validated our approach using a custom microarray, using sequences identified as DNA fingerprints of Bacillus anthracis. Hybridization results showed that the sequences found using our algorithm were truly unique to B. anthracis and were able to distinguish B. anthracis from its close relatives B. cereus and B. thuringiensis.
Detection of Viral RNA From Paraffin-Embedded Tissues After Prolonged Formalin Fixation ( Book )
1 edition published in 2009 in English and held by 1 library worldwide
Isolating amplifiable RNA from formalin-fixed, paraffin-embedded (FFPE) tissues is more difficult than isolating DNA because the potential for RNA degradation by RNases, chemical modification of the RNA by addition of methylol groups ( -CH2OH), and cross-linking of nucleic acids and proteins during the fixation process. In our laboratories, these difficulties are increased when extended fixation times of 3 to 4 weeks are required for tissues infected with BSL-3 or -4 agents. Four commercially available kits with different nucleic acid isolation chemistries were evaluated for their ability to isolate RNA from FFPE West Nile virus-infected tissues. The quality of the extracted RNA was determined by using a fluorogenic 5' nuclease TaqManTM) PCR assay. A modification of the Paraffin Block RNA Isolation Kit that included an overnight proteinase K digestion was necessary to obtain amplifiable RNA from tissues formalin-fixed for 21 days. Extracting TaqManTM amplifiable RNA from Marburg- and Ebola virus-infected tissues, formalin-fixed for at least 30 days, further tested the modified extraction method. This improved extraction procedure for obtaining amplifiable RNA combined with the more sensitive and specific fluorogenic probe-based PCR assays will now permit retrospective and prospective studies on FFPE tissues infected with BSL-3 and -4 pathogens.
Cynomolgus Macaque as an Animal Model for Severe Acute Respiratory Syndrome ( Book )
1 edition published in 2006 in English and held by 1 library worldwide
The emergence of severe acute respiratory syndrome (SARS) in 2002 - 2003 had a tremendous global impact. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. In order to characterize clinically relevant parameters of SARS-CoV infection in non-human primates, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva and Group II intravenously. Animals in Groups I and II developed mild-moderate symptomatic illness. All animals demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in Groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 -10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more significant disease that intravenous inoculation. SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of human cases of SARS; however, our results suggest similarities to the more mild syndrome of SARS infection characteristically seen in young children.
Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.I.D., the LightCycler, and the Smart Cycler Platforms ( Book )
1 edition published in 2006 in English and held by 1 library worldwide
Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for the molecular identification of these agents. A common chemistry that can be used on a variety of rapid, real-time PCR instruments provides the greatest flexibility for assay utilization. Methods: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by using Idaho Technology, Inc. buffers and dNTPs supplemented with Invitrogen Platinum Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the Idaho Technology, Inc. R.A.P.I.D., the Roche LightCycler, and the Cepheid Smart Cycler . Results: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. Conclusion: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, a common chemistry could be used for DNA templates that resulted in similar performance, sensitivity, and specificity on all three platforms.
Monkeypox detection in rodents using real-time 3'minor groove binder Taqman assays on the Roche LightCycler, Laboratory Investigation 84:1200-1208 ( Book )
1 edition published in 2004 in English and held by 1 library worldwide
During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses.
Smallpox and pan-Orthodox Virus Detection by Real-Time 3'-Minor Groove Binder TaqMan Assays Oil the Roche LightCycler and the Cepheid Smart Cycler Platforms ( Book )
1 edition published in 2003 in English and held by 1 library worldwide
We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs.