Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

High-quality nucleic acids are critical for optimal PCR-based diagnostics and pathogen detection. Rapid sample processing time is important for the earliest administration of therapeutic and containment measures, especially in the case of biothreat agents. In this context, we compared the Fujifilm QuickGene-Mini80 to Qiagen\u27s QIAamp Mini Purification kits for extraction of DNA and RNA for potential use in austere settings. Qiagen (QIAamp) column-based extraction is the currently recommended purification platform by United States Army Medical Research Institute for Infectious Diseases for both DNA and RNA extraction. However, this sample processing system requires dedicated laboratory equipment including a centrifuge. In this study, we investigated the QuickGene-Mini80, which does not require centrifugation, as a suitable platform for nucleic acid extraction for use in resource-limited locations. Quality of the sample extraction was evaluated using pathogen-specific, real-time PCR assays for nucleic acids extracted from viable and γ-irradiated Bacillus anthracis, Yersinia pestis, vaccinia virus, Venezuelan equine encephalitis virus, or B. anthracis spores in buffer or human whole blood. QuickGene-Mini80 and QIAamp performed similarly for DNA extraction regardless of organism viability. It was noteworthy that γ-irradiation did not have a significant impact on real-time PCR for organism detection. Comparison with QIAamp showed a less than adequate performance of the Fujifilm instrument for RNA extraction. However, QuickGene-Mini80 remains a viable alternative to QIAamp for DNA extraction for use in remote settings due to extraction quality, time efficiency, reduced instrument requirements, and ease of use

Cynomolgus Macaque as an Animal Model for Severe Acute Respiratory Syndrome

BACKGROUND: The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV. METHODS AND FINDINGS: In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, 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 III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs 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 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain. CONCLUSIONS: SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children

Identification of genomic signatures for the design of assays for the detection and monitoring of anthrax threats

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. 1

Identification of genomic signatures for the design of assays for the detection and monitoring of anthrax threats

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. 1

Detection of the Bacillus anthracis gyrA Gene by Using a Minor Groove Binder Probe

Identification of chromosomal markers for rapid detection of Bacillus anthracis is difficult because significant chromosomal homology exists among B. anthracis, Bacillus cereus, and Bacillus thuringiensis. We evaluated the bacterial gyrA gene as a potential chromosomal marker for B. anthracis. A real-time PCR assay was developed for the detection of B. anthracis. After analysis of the unique nucleotide sequence of the B. anthracis gyrA gene, a fluorescent 3′ minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102 Bacillus strains. The assay was found to be specific for all 43 strains of B. anthracis tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the gyrA gene as a specific chromosomal marker for B. anthracis

Real-Time PCR Assay To Detect Smallpox Virus

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence