RESUMEN
FDA proactively invests in tools to support innovation of emerging technologies, such as infectious disease next generation sequencing (ID-NGS). Here, we introduce FDA-ARGOS quality-controlled reference genomes as a public database for diagnostic purposes and demonstrate its utility on the example of two use cases. We provide quality control metrics for the FDA-ARGOS genomic database resource and outline the need for genome quality gap filling in the public domain. In the first use case, we show more accurate microbial identification of Enterococcus avium from metagenomic samples with FDA-ARGOS reference genomes compared to non-curated GenBank genomes. In the second use case, we demonstrate the utility of FDA-ARGOS reference genomes for Ebola virus target sequence comparison as part of a composite validation strategy for ID-NGS diagnostic tests. The use of FDA-ARGOS as an in silico target sequence comparator tool combined with representative clinical testing could reduce the burden for completing ID-NGS clinical trials.
Asunto(s)
Enfermedades Transmisibles/diagnóstico , Bases de Datos de Ácidos Nucleicos/normas , Genoma , Acceso a la Información , Enfermedades Transmisibles/microbiología , Bases de Datos de Ácidos Nucleicos/organización & administración , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to determine its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.
Asunto(s)
Análisis por Micromatrices/métodos , Microbiota , Animales , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Heces/microbiología , Heces/virología , Genoma Bacteriano , Genoma Viral , Ensayos Analíticos de Alto Rendimiento , Hibridación de Ácido Nucleico , Poxviridae/genética , Poxviridae/aislamiento & purificación , Reproducibilidad de los Resultados , Shigella flexneri/genética , Shigella flexneri/aislamiento & purificación , PorcinosRESUMEN
A guanine mononucleotide repeat in the rpsA (tp0279) gene was evaluated for improved strain discrimination using 72 Treponema pallidum-positive specimens. The tandem repeat combined with the enhanced Centers for Disease Control and Prevention typing system resulted in increased discrimination and should be useful for molecular epidemiologic studies on syphilis especially in outbreaks and among men who have sex with men.
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ADN Bacteriano/genética , Tipificación Molecular/métodos , Sífilis/microbiología , Secuencias Repetidas en Tándem , Treponema pallidum/clasificación , Genotipo , Homosexualidad Masculina , Humanos , Masculino , Mutación Puntual , ARN Ribosómico 23S/genéticaRESUMEN
An estimated 1.5 billion microbial infections occur globally each year and result in â¼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of â¼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE: This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.
Asunto(s)
Infecciones Bacterianas/diagnóstico , Infección Hospitalaria/diagnóstico , ADN Bacteriano/análisis , Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Sistemas de Atención de Punto , Acinetobacter baumannii/clasificación , Acinetobacter baumannii/genética , Infecciones Bacterianas/microbiología , Infección Hospitalaria/microbiología , Cartilla de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Enterobacter/clasificación , Enterobacter/genética , Enterococcus faecium/clasificación , Enterococcus faecium/genética , Humanos , Klebsiella pneumoniae/clasificación , Klebsiella pneumoniae/genética , Microfluídica/instrumentación , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/genética , Staphylococcus aureus/clasificación , Staphylococcus aureus/genéticaRESUMEN
The past decade has seen considerable development in the diagnostic application of nonculture methods, including nucleic acid amplification-based methods and mass spectrometry, for the diagnosis of infectious diseases. The implications of these new culture-independent diagnostic tests (CIDTs) include bypassing the need to culture organisms, thus potentially affecting public health surveillance systems, which continue to use isolates as the basis of their surveillance programs and to assess phenotypic resistance to antimicrobial agents. CIDTs may also affect the way public health practitioners detect and respond to a bioterrorism event. In response to a request from the Department of Homeland Security, Los Alamos National Laboratory and the Centers for Disease Control and Prevention cosponsored a workshop to review the impact of CIDTs on the rapid detection and identification of biothreat agents. Four panel discussions were held that covered nucleic acid amplification-based diagnostics, mass spectrometry, antibody-based diagnostics, and next-generation sequencing. Exploiting the extensive expertise available at this workshop, we identified the key features, benefits, and limitations of the various CIDT methods for providing rapid pathogen identification that are critical to the response and mitigation of a bioterrorism event. After the workshop we conducted a thorough review of the literature, investigating the current state of these 4 culture-independent diagnostic methods. This article combines information from the literature review and the insights obtained at the workshop.
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Biovigilancia/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoensayo/métodos , Espectrometría de Masas/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Vigilancia en Salud Pública/métodos , HumanosRESUMEN
BACKGROUND: Historically, identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes. However, these methods can be limiting: Even highly sensitive PCR-based assays must be continually updated due to signature degradation as new target strains and near neighbors are sequenced. Thus, there has been a need for assays that do not suffer as greatly from these limitations and/or biases. Recent advances in library preparation technologies for Next-Generation Sequencing (NGS) are focusing on the use of targeted amplification and targeted enrichment/capture to ensure that the most highly discriminating regions of the genomes of known targets (organism-unique regions and/or regions containing functionally important genes or phylogenetically-discriminating SNPs) will be sequenced, regardless of the complex sample background. RESULTS: In the present study, we have assessed the feasibility of targeted sequence enhancement via amplification to facilitate detection of a bacterial pathogen present in low copy numbers in a background of human genomic material. Our results indicate that the targeted amplification of signature regions can effectively identify pathogen genomic material present in as little as 10 copies per ml in a complex sample. Importantly, the correct species and strain calls could be made in amplified samples, while this was not possible in unamplified samples. CONCLUSIONS: The results presented here demonstrate the efficacy of a targeted amplification approach to biothreat detection, using multiple highly-discriminative amplicons per biothreat organism that provide redundancy in case of variation in some primer regions. Importantly, strain level discrimination was possible at levels of 10 genome equivalents. Similar results could be obtained through use of panels focused on the identification of amplicons targeted for specific genes or SNPs instead of, or in addition to, those targeted for specific organisms (ongoing gene-targeting work to be reported later). Note that without some form of targeted enhancement, the enormous background present in complex clinical and environmental samples makes it highly unlikely that sufficient coverage of key pathogen(s) present in the sample will be achieved with current NGS technology to guarantee that the most highly discriminating regions will be sequenced.
Asunto(s)
Biblioteca de Genes , Genoma Bacteriano/genética , Genoma Humano/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/métodos , HumanosRESUMEN
The number and prevalence of diseases is rapidly increasing in the marine ecosystem. Although there is an increase in the number of marine diseases observed world-wide, current understanding of the pathogens associated with marine mammals is limited. An important need exists to develop and apply platforms for rapid detection and characterization of pathogenic agents to assess, prevent and respond to disease outbreaks. In this study, a broad-spectrum molecular detection technology capable of detecting all sequenced microbial organisms, the Lawrence Livermore Microbial Detection Array, was used to assess the microbial agents that could be associated with wild Atlantic dolphins. Blowhole, gastric, and fecal samples from 8 bottlenose dolphins were collected in Charleston, SC, as part of the dolphin assessment effort. The array detected various microbial agents from the dolphin samples. Clostridium perfringens was most prevalent in the samples surveyed using the microarray. This pathogen was also detected using microbiological culture techniques. Additionally, Campylobacter sp., Staphylococcus sp., Erwinia amylovora, Helicobacter pylori, and Frankia sp. were also detected in more than one dolphin using the microarray, but not in culture. This study provides the first survey of pathogens associated with 3 tissue types in dolphins using a broad-spectrum microbial detection microarray and expands insight on the microbial community profile in dolphins.
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Animales Salvajes , Bacterias/aislamiento & purificación , Infecciones Bacterianas/veterinaria , Delfín Mular/microbiología , Animales , Bacterias/clasificación , Infecciones Bacterianas/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinariaRESUMEN
Identifying causative disease agents in human patients from shotgun metagenomic sequencing (SMS) presents a powerful tool to apply when other targeted diagnostics fail. Numerous technical challenges remain, however, before SMS can move beyond the role of research tool. Accurately separating the known and unknown organism content remains difficult, particularly when SMS is applied as a last resort. The true amount of human DNA that remains in a sample after screening against the human reference genome and filtering nonbiological components left from library preparation has previously been underreported. In this study, we create the most comprehensive collection of microbial and reference-free human genetic variation available in a database optimized for efficient metagenomic search by extracting sequences from GenBank and the 1000 Genomes Project. The results reveal new human sequences found in individual Human Microbiome Project (HMP) samples. Individual samples contain up to 95% human sequence, and 4% of the individual HMP samples contain 10% or more human reads. Left unidentified, human reads can complicate and slow down further analysis and lead to inaccurately labeled microbial taxa and ultimately lead to privacy concerns as more human genome data is collected.
Asunto(s)
Genoma Microbiano , Metagenoma , Metagenómica/métodos , Microbiota , Biología Computacional/métodos , Bases de Datos de Ácidos Nucleicos , Humanos , Curva ROCRESUMEN
UNLABELLED: We announce the release of kSNP3.0, a program for SNP identification and phylogenetic analysis without genome alignment or the requirement for reference genomes. kSNP3.0 is a significantly improved version of kSNP v2. AVAILABILITY AND IMPLEMENTATION: kSNP3.0 is implemented as a package of stand-alone executables for Linux and Mac OS X under the open-source BSD license. The executable packages, source code and a full User Guide are freely available at https://sourceforge.net/projects/ksnp/files/ CONTACT: barryghall@gmail.com.
Asunto(s)
Biología Computacional/métodos , Escherichia coli/genética , Genoma Bacteriano , Filogenia , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Bases de Datos de Ácidos Nucleicos , Escherichia coli/clasificación , Anotación de Secuencia MolecularRESUMEN
Accurate and sensitive detection of microbes against a complex background is a problem common to multiple aspects of human health, such as vaccines and other biologicals safety, blood safety, and diagnosing infectious diseases in humans or other hosts. The microbes in question could be bacterial, viral, fungal, or parasitical. To defend against such a broad array of microbes of potential safety concern, we need more than single-target polymerase chain reaction (PCR) assays. Technologies such as highly-multiplexed PCR, broad-spectrum DNA/RNA microarrays, and next-generation sequencing are all potentiallycapable to provide increased protection against microbial contamination. Regulatory processes are currently struggling to keep up with rapid advances in all of these technologies, each of which is firmly based upon nucleic acid sequencing resulting in generation of megabases of data. A major question is the level of quality required for genomic data and metadata for the reference databases that are needed to allow these technologies to be developed, validated, and then used for front-line protection of human health. The background of this general problem is discussed and one example of an ongoing effort to establish quality levels for a bacterial genome reference database is presented.
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Bacterias/genética , Técnicas Bacteriológicas/normas , ADN Bacteriano/genética , Bases de Datos Genéticas/normas , Genoma Bacteriano , Bacterias/clasificación , Técnicas Bacteriológicas/tendencias , Exactitud de los Datos , Minería de Datos , Bases de Datos Genéticas/tendencias , Difusión de Innovaciones , Predicción , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Reacción en Cadena de la Polimerasa Multiplex/normas , Filogenia , Control de CalidadRESUMEN
BACKGROUND: Pairing up primers to amplify desired targets and avoid undesired cross reactions can be a combinatorial challenge. Effective prediction of specificity and inclusivity from multiplexed primers and TaqMan®/Luminex® probes is a critical step in PCR design. RESULTS: Code is described to identify all primer and probe combinations from a list of unpaired, unordered candidates that should produce a product. It predicts and extracts all amplicon sequences in a large sequence database from a list of primers and probes, allowing degenerate bases and user-specified levels of primer-target mismatch tolerance. Amplicons hit by TaqMan®/Luminex® probes are indicated, and products may be annotated with gene information from NCBI. Fragment length distributions are calculated to predict electrophoretic gel banding patterns. CONCLUSIONS: Simulate_PCR is the only freely available software that can be run from the command line for high throughput applications which can calculate all products from large lists of primers and probes compared to a large sequence database such as nt. It requires no prior knowledge of how primers should be paired. Degenerate bases are allowed and entire amplicon sequences are extracted and annotated with gene information. Examples are provided for sets of TaqMan®/Luminex® PCR signatures predicted to amplify all HIV-1 genomes, all Coronaviridae genomes, and a group of antibiotic resistance genes. The software is a command line perl script freely available as open source.
Asunto(s)
Biología Computacional/métodos , Cartilla de ADN/genética , Sondas de ADN/genética , Anotación de Secuencia Molecular , Programas Informáticos , Coronaviridae/genética , Farmacorresistencia Microbiana/genética , VIH-1/genética , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1h. The LLMDA was able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 µL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.
Asunto(s)
Bacterias/aislamiento & purificación , Análisis por Micromatrices/métodos , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Virus/aislamiento & purificación , Bacterias/clasificación , Bacterias/genética , Humanos , Sensibilidad y Especificidad , Manejo de Especímenes/métodos , Virus/clasificación , Virus/genéticaRESUMEN
Microarrays to characterize single nucleotide polymorphisms (SNPs) provide a cost-effective and rapid method (under 24h) to genotype microbes as an alternative to sequencing. We developed a pipeline for SNP discovery and microarray design that scales to 100's of microbial genomes. Here we tested various SNP probe design strategies against 8 sequenced isolates of Bacillus anthracis to compare sequence and microarray data. The best strategy allowed probe length to vary within 32-40 bp to equalize hybridization free energy. This strategy resulted in a call rate of 99.52% and concordance rate of 99.86% for finished genomes. Other probe design strategies averaged substantially lower call rates (94.65-96.41%) and slightly lower concordance rates (99.64-99.80%). These rates were lower for draft than finished genomes, consistent with higher incidence of sequencing errors and gaps. Highly accurate SNP calls were possible in complex soil and blood backgrounds down to 1000 copies, and moderately accurate SNP calls down to 100 spiked copies. The closest genome to the spiked strain was correctly identified at only 10 spiked copies. Discrepancies between sequence and array data did not alter the SNP-based phylogeny, regardless of the probe design strategy, indicating that SNP arrays can accurately place unsequenced isolates on a phylogeny.
Asunto(s)
ADN Bacteriano/análisis , Técnicas de Genotipaje/métodos , Tipificación Molecular/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Bacillus anthracis/clasificación , Bacillus anthracis/genética , Sondas de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Modelos Genéticos , FilogeniaRESUMEN
BACKGROUND: High throughput sequencing is beginning to make a transformative impact in the area of viral evolution. Deep sequencing has the potential to reveal the mutant spectrum within a viral sample at high resolution, thus enabling the close examination of viral mutational dynamics both within- and between-hosts. The challenge however, is to accurately model the errors in the sequencing data and differentiate real viral mutations, particularly those that exist at low frequencies, from sequencing errors. RESULTS: We demonstrate that overlapping read pairs (ORP) -- generated by combining short fragment sequencing libraries and longer sequencing reads -- significantly reduce sequencing error rates and improve rare variant detection accuracy. Using this sequencing protocol and an error model optimized for variant detection, we are able to capture a large number of genetic mutations present within a viral population at ultra-low frequency levels (<0.05%). CONCLUSIONS: Our rare variant detection strategies have important implications beyond viral evolution and can be applied to any basic and clinical research area that requires the identification of rare mutations.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Virus/genética , Benchmarking , Genoma Viral/genética , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Lassa hemorrhagic fever (LHF) is a rodent-borne viral disease that can be fatal for human beings. In this study, an attenuated Lassa vaccine candidate, ML29, was tested in SIV-infected rhesus macaques for its ability to elicit immune responses without instigating signs pathognomonic for arenavirus disease. ML29 is a reassortant between Lassa and Mopeia viruses that causes a transient infection in non-human primates and confers sterilizing protection from lethal Lassa viral challenge. However, since the LHF endemic area of West Africa also has high HIV seroprevalence, it is important to determine whether vaccination could be safe in the context of HIV infection. RESULTS: SIV-infected and uninfected rhesus macaques were vaccinated with the ML29 virus and monitored for specific humoral and cellular immune responses, as well as for classical and non-classical signs of arenavirus disease. Classical disease signs included viremia, rash, respiratory distress, malaise, high liver enzyme levels, and virus invasion of the central nervous system. Non-classical signs, derived from profiling the blood transcriptome of virulent and non-virulent arenavirus infections, included increased expression of interferon-stimulated genes (ISG) and decreased expression of COX2, IL-1ß, coagulation intermediates and nuclear receptors needed for stress signaling. All vaccinated monkeys showed ML29-specific antibody responses and ML29-specific cell-mediated immunity. CONCLUSION: SIV-infected and uninfected rhesus macaques responded similarly to ML29 vaccination, and none developed chronic arenavirus infection. Importantly, none of the macaques developed signs, classical or non-classical, of arenavirus disease.
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Coinfección/inmunología , Infecciones por VIH/inmunología , Fiebre de Lassa/prevención & control , Virus Lassa/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Coinfección/prevención & control , Coinfección/virología , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , Humanos , Fiebre de Lassa/complicaciones , Fiebre de Lassa/inmunología , Fiebre de Lassa/virología , Macaca mulatta , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were "selected" from a pre-existing pool rather than through de novo mutation and subsequent population fixation.
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Bovinos/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Coronavirus Bovino/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia de Consenso , Coronavirus Bovino/química , Coronavirus Bovino/fisiología , Variación Genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Tasa de Mutación , Filogenia , Mutación Puntual , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genética , Internalización del VirusRESUMEN
CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) We designed the Lawrence Livermore Microbial Detection Array (LLMDA), which contains 388,000 DNA probes. This array can detect any sequenced viruses or bacteria within 24 h. In addition, the oligonucleotide probes were selected to enable detection of novel, divergent species with homology to sequenced organisms. We recently used this array to identify an adventitious virus from a vaccine product. We have also used this array to detect viral and bacterial infections from various human clinical samples. Broad-spectrum microbial detection microarrays are efficient and cost-effective tools to rapidly screen cell bank samples, raw materials, vaccine samples, and clinical samples to ensure drug, food, and health safety in the United States and worldwide.
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Análisis de Secuencia por Matrices de Oligonucleótidos , Virus , Bacterias/genética , Infecciones Bacterianas/microbiología , Sondas de ADN , Humanos , Sondas de Oligonucleótidos/genética , Estados Unidos , United States Food and Drug Administration , Virus/genéticaRESUMEN
BACKGROUND: Identifying the bacteria and viruses present in a complex sample is useful in disease diagnostics, product safety, environmental characterization, and research. Array-based methods have proven utility to detect in a single assay at a reasonable cost any microbe from the thousands that have been sequenced. METHODS: We designed a pan-Microbial Detection Array (MDA) to detect all known viruses (including phages), bacteria and plasmids and developed a novel statistical analysis method to identify mixtures of organisms from complex samples hybridized to the array. The array has broader coverage of bacterial and viral targets and is based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms, and to have no significant matches to the human genome sequence. RESULTS: In blinded testing on spiked samples with single or multiple viruses, the MDA was able to correctly identify species or strains. In clinical fecal, serum, and respiratory samples, the MDA was able to detect and characterize multiple viruses, phage, and bacteria in a sample to the family and species level, as confirmed by PCR. CONCLUSIONS: The MDA can be used to identify the suite of viruses and bacteria present in complex samples.
Asunto(s)
Bacterias/aislamiento & purificación , Análisis por Micromatrices/métodos , Virus/aislamiento & purificación , Algoritmos , Animales , Bacterias/genética , Bovinos , Sondas de ADN/metabolismo , Entropía , Heces/microbiología , Heces/virología , Humanos , Funciones de Verosimilitud , Hibridación de Ácido Nucleico , Esputo/microbiología , Esputo/virología , Virus/genéticaRESUMEN
BACKGROUND: Finding the amino acid mutations that affect the severity of influenza infections remains an open and challenging problem. Of special interest is better understanding how current circulating influenza strains could evolve into a new pandemic strain. Influenza proteomes from distinct viral phenotype classes were searched for class specific amino acid mutations conserved in past pandemics, using reverse engineered linear classifiers. RESULTS: Thirty-four amino acid markers associated with host specificity and high mortality rate were found. Some markers had little impact on distinguishing the functional classes by themselves, however in combination with other mutations they improved class prediction. Pairwise combinations of influenza genomes were checked for reassortment and mutation events needed to acquire the pandemic conserved markers. Evolutionary pathways involving H1N1 human and swine strains mixed with avian strains show the potential to acquire the pandemic markers with a double reassortment and one or two amino acid mutations. CONCLUSION: The small mutation combinations found at multiple protein positions associated with viral phenotype indicate that surveillance tools could monitor genetic variation beyond single point mutations to track influenza strains. Finding that certain strain combinations have the potential to acquire pandemic conserved markers through a limited number of reassortment and mutation events illustrates the potential for reassortment and mutation events to lead to new circulating influenza strains.
Asunto(s)
Evolución Molecular , Marcadores Genéticos , Genoma Viral , Subtipo H5N1 del Virus de la Influenza A/genética , Proteómica , Secuencia de Aminoácidos , Animales , Aves/virología , Secuencia Conservada , Brotes de Enfermedades , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Gripe Aviar/virología , Gripe Humana/epidemiología , Gripe Humana/virología , Mutación , Virus Reordenados/genética , Alineación de Secuencia , Especificidad de la Especie , Porcinos , Proteínas Virales/genéticaRESUMEN
Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.