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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.
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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
Pregnancy complications are poorly represented in the archeological record, despite their importance in contemporary and ancient societies. While excavating a Byzantine cemetery in Troy, we discovered calcified abscesses among a woman's remains. Scanning electron microscopy of the tissue revealed 'ghost cells', resulting from dystrophic calcification, which preserved ancient maternal, fetal and bacterial DNA of a severe infection, likely chorioamnionitis. Gardnerella vaginalis and Staphylococcus saprophyticus dominated the abscesses. Phylogenomic analyses of ancient, historical, and contemporary data showed that G. vaginalis Troy fell within contemporary genetic diversity, whereas S. saprophyticus Troy belongs to a lineage that does not appear to be commonly associated with human disease today. We speculate that the ecology of S. saprophyticus infection may have differed in the ancient world as a result of close contacts between humans and domesticated animals. These results highlight the complex and dynamic interactions with our microbial milieu that underlie severe maternal infections.
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Absceso/patología , Fósiles , Infecciones por Bacterias Grampositivas/patología , Complicaciones Infecciosas del Embarazo/patología , Absceso/microbiología , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Femenino , Gardnerella vaginalis/clasificación , Gardnerella vaginalis/genética , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Microscopía Electrónica de Rastreo , Embarazo , Staphylococcus saprophyticus/clasificación , Staphylococcus saprophyticus/genéticaRESUMEN
Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30-40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease.
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Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.
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Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broad panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Finally, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.
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Virus de la Encefalitis Equina Venezolana/genética , Filogenia , Polimorfismo de Nucleótido Simple , Animales , Cricetinae/virología , Culicidae/virología , Virus de la Encefalitis Equina Venezolana/aislamiento & purificación , Encefalomielitis Equina Venezolana/epidemiología , Variación Genética , Genoma Viral , Genotipo , Interacciones Huésped-Patógeno/genética , México/epidemiología , Ratones Endogámicos/virología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , FenotipoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0146251.].
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Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater than 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.
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Infecciones por Coronavirus/genética , Infecciones por Coronavirus/transmisión , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Mutación , Animales , Camelus , Chlorocebus aethiops , Humanos , Células VeroRESUMEN
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.
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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.
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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.
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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
Many of the disease syndromes challenging the commercial swine industry involve the analysis of complex problems caused by polymicrobial, emerging or reemerging, and transboundary pathogens. This study investigated the utility of the Lawrence Livermore Microbial Detection Array (Lawrence Livermore National Laboratory, Livermore, California), designed to detect 8,101 species of microbes, in the evaluation of known and unknown microbes in serum, oral fluid, and tonsil from pigs experimentally coinfected with Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus-2 (PCV-2). The array easily identified PRRSV and PCV-2, but at decreased sensitivities compared to standard polymerase chain reaction detection methods. The oral fluid sample was the most informative, possessing additional signatures for several swine-associated bacteria, including Streptococcus sp., Clostridium sp., and Staphylococcus sp.
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Infecciones por Circoviridae/diagnóstico , Circovirus/aislamiento & purificación , Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificación , Animales , California , Infecciones por Circoviridae/sangre , Infecciones por Circoviridae/virología , Circovirus/genética , Coinfección , Femenino , Masculino , Tonsila Palatina/microbiología , Tonsila Palatina/virología , Reacción en Cadena de la Polimerasa/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/sangre , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Valor Predictivo de las Pruebas , Saliva/microbiología , Saliva/virología , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/virologíaRESUMEN
Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.
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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.
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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
Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.
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Técnicas de Diagnóstico Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Infecciones por Virus ARN/diagnóstico , Virus ARN/clasificación , Virus ARN/aislamiento & purificación , Biomarcadores/metabolismo , ADN Viral/genética , Perfilación de la Expresión Génica , Humanos , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/virología , Virus ARN/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Manejo de EspecímenesRESUMEN
Vaccinations against the encephalitic alphaviruses (western, eastern, and Venezuelan equine encephalitis virus) are of significant interest to biological defense, public health, and agricultural communities alike. Although vaccines licensed for veterinary applications are used in the Western Hemisphere and attenuated or inactivated viruses have been used under Investigational New Drug status to protect at-risk personnel, there are currently no licensed vaccines for use in humans. Here, we will discuss the need for a trivalent vaccine that can protect humans against all three viruses, recent progress to such a vaccine, and a strategy to continue development to Food and Drug Administration licensure.
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Infecciones por Alphavirus/prevención & control , Alphavirus/inmunología , Encefalitis Viral/prevención & control , Vacunas Virales , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina del Este/inmunología , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/inmunología , Encefalitis Viral/virología , Humanos , Alineación de Secuencia , VacunaciónRESUMEN
Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.
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Bacterias/clasificación , Bacterias/aislamiento & purificación , Biota , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis por Micromatrices/métodos , Infección de Heridas/microbiología , Adulto , Bacterias/genética , Carga Bacteriana , Humanos , Personal Militar , Cicatrización de Heridas , Adulto JovenRESUMEN
In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technology was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15-62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae, Bacteroidaceae, and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.
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Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis ("Black Death" plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.
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Arqueología , ADN Bacteriano , Análisis de Secuencia por Matrices de Oligonucleótidos , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Filogenia , Vibrio cholerae/clasificación , Vibrio cholerae/genética , Yersinia pestis/clasificación , Yersinia pestis/genéticaRESUMEN
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.