RESUMO
BACKGROUND: Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). RESULTS: A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. CONCLUSIONS: The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.
Assuntos
Contaminação por DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , DNA/análise , Óxido de Etileno/farmacologia , Genoma Bacteriano , Genoma Humano , Humanos , Indicadores e Reagentes , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
Molecular bioforensic research is dependent on rapid and sensitive methods such as real-time PCR (qPCR) for the identification of microorganisms. The use of synthetic positive control templates containing small modifications outside the primer and probe regions is essential to ensure all aspects of the assay are functioning properly, including the primers and probes. However, a typical qPCR or reverse transcriptase qPCR (qRT-PCR) assay is limited in differentiating products generated from positive controls and biological samples because the fluorescent probe signals generated from each type of amplicon are indistinguishable. Additional methods used to differentiate amplicons, including melt curves, secondary probes, and amplicon sequencing, require significant time to implement and validate and present technical challenges that limit their use for microbial forensic applications. To solve this problem, we have developed a novel application of electrospray ionization mass spectrometry (ESI-MS) to rapidly differentiate qPCR amplicons generated with positive biological samples from those generated with synthetic positive controls. The method has sensitivity equivalent to qPCR and supports the confident and timely determination of the presence of a biothreat agent that is crucial for policymakers and law enforcement. Additionally, it eliminates the need for time-consuming methods to confirm qPCR results, including development and validation of secondary probes or sequencing of small amplicons. In this study, we demonstrate the effectiveness of this approach with microbial forensic qPCR assays targeting multiple biodefense agents (bacterial, viral, and toxin) for the ability to rapidly discriminate between a positive control and a positive sample.
Assuntos
Bioterrorismo/prevenção & controle , DNA Bacteriano/análise , Ciências Forenses/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Espectrometria de Massas por Ionização por Electrospray , Clostridium botulinum tipo F/genética , Bactérias Gram-Negativas/genética , Vírus Hendra/genética , Vírus Nipah/genética , Polimorfismo de Nucleotídeo Único , Sensibilidade e EspecificidadeRESUMO
Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.
Assuntos
Bactérias/genética , Armas Biológicas , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Vírus/genética , Bactérias/isolamento & purificação , Bioensaio , Análise por Conglomerados , Primers do DNA/metabolismo , Reações Falso-Negativas , Limite de Detecção , Relatório de Pesquisa , Sensibilidade e Especificidade , Estatística como Assunto , Vírus/isolamento & purificaçãoRESUMO
OBJECTIVE: To develop and evaluate a rapid and accurate assay involving PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples for the detection of Dirofilaria immitis infection in dogs. SAMPLE: Whole blood nucleic acid extracts from 29 dogs experimentally infected with D immitis (and in which circulating D immitis antigen was detected) and 10 uninfected dogs. PROCEDURES: 16 of the 29 whole blood samples from infected dogs were examined at the time of collection for circulating microfilaria. Nucleic acids were extracted from all whole blood specimens and underwent PCR amplification with 12 PCR primer pairs designed to detect a wide range of pathogens (including the Wolbachia endosymbiont of D immitis) and electrospray ionization mass spectrometry. RESULTS: On the basis of assay results, heartworm infection was detected in 13 of 13 antigen-positive dogs of unknown microfilaria status, 11 of 11 antigen-positive dogs with circulating microfilaria, 0 of 3 antigen-positive dogs tested at 3 months after larval infection, 0 of 2 antigen-positive dogs with occult infections, and 0 of 10 uninfected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: With the assay under investigation, it was possible to identify D immitis infection in dogs with circulating microfilaria via detection of the obligate Wolbachia endosymbiont of D immitis. It was not possible to identify dogs with occult infections, which suggested that circulating microfilaria must be present to detect infection with this assay, although further studies would be required to verify that finding.
Assuntos
Dirofilariose/diagnóstico , Doenças do Cão/diagnóstico , Ácidos Nucleicos/sangue , Animais , Sequência de Bases , Primers do DNA/genética , DNA Ribossômico/genética , Dirofilariose/genética , Doenças do Cão/genética , Cães , Espectrometria de Massas/métodos , Espectrometria de Massas/veterinária , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA/veterináriaRESUMO
Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.
Assuntos
Alelos , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Espectrometria de Massas por Ionização por Electrospray/métodos , Automação , Sequência de Bases , Primers do DNA , Eletroforese Capilar , Humanos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Homologia de Sequência do Ácido NucleicoRESUMO
Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Mycobacterium/classificação , Mycobacterium/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Técnicas Bacteriológicas/métodos , Primers do DNA/genética , República da Geórgia , Humanos , Isoniazida/farmacologia , Mycobacterium/isolamento & purificação , Cidade de Nova Iorque , Rifampina/farmacologia , África do Sul , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs. We then focus on a new method that combines nucleic acid amplification with mass spectrometry in a novel approach to molecular diagnosis of bloodstream infections.
Assuntos
Bacteriemia/diagnóstico , DNA Bacteriano/sangue , DNA Fúngico/sangue , Fungemia/diagnóstico , Técnicas de Diagnóstico Molecular , Idoso , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto JovemRESUMO
A gram-negative, rod-shaped microorganism was detected in a 69-year-old man suffering from chronic back pain but otherwise exhibiting no signs of infection. The bacterium could not be identified using any routine diagnostic modality. A research use only application utilizing PCR and Mass Spectrometry was performed on nucleic acid extracted from the tissue sample. These studies resulted in the implication of Bartonella quintana as the underlying cause of the infection. B. quintana is not a well-known cause of an abdominal aortic mycotic aneurysm. This article will discuss the B. quintana infection, its diagnosis and treatment, and reinforce the potential of B. quintana as a possible etiology in mycotic aneurysms that show no apparent indications of infection. It will also explore the potential use of polymerase chain reaction detected by electrospray ionization mass spectrometry (PCR/ESI-MS) to help identify B. quintana in a situation where other conventional methods prove non-informative.
Assuntos
Aneurisma Infectado/microbiologia , Aorta Abdominal/microbiologia , Aneurisma da Aorta Abdominal/microbiologia , Bartonella quintana/isolamento & purificação , Febre das Trincheiras/diagnóstico , Idoso , Aneurisma Infectado/diagnóstico , Aneurisma da Aorta Abdominal/diagnóstico , Humanos , Masculino , Fatores de Tempo , Febre das Trincheiras/microbiologiaRESUMO
We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.
Assuntos
DNA Mitocondrial/química , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Composição de Bases , Bases de Dados Genéticas , Genética Forense , HumanosRESUMO
The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.
Assuntos
Espectrometria de Massas/métodos , Orthopoxvirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Primers do DNA , DNA Viral/genética , Humanos , Dados de Sequência Molecular , Orthopoxvirus/genética , Coelhos , Homologia de Sequência do Ácido NucleicoRESUMO
We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.
Assuntos
Infecção Hospitalar/prevenção & controle , Genética Microbiana/métodos , Epidemiologia Molecular/métodos , Vigilância da População/métodos , Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Surtos de Doenças , Genes Bacterianos , Genótipo , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas por Ionização por Electrospray/métodos , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
We describe a new technology, the Ibis T5000, for the identification of pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic acid amplification to high-performance electrospray ionization mass spectrometry and base-composition analysis. The system enables the identification and quantification of a broad set of pathogens, including all known bacteria, all major groups of pathogenic fungi and the major families of viruses that cause disease in humans and animals, along with the detection of virulence factors and antibiotic resistance markers.
Assuntos
Técnicas Biossensoriais/métodos , Doenças Transmissíveis Emergentes/diagnóstico , Técnicas Microbiológicas , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Controle de Doenças Transmissíveis/métodos , Doenças Transmissíveis Emergentes/microbiologia , Doenças Transmissíveis Emergentes/parasitologia , Doenças Transmissíveis Emergentes/virologia , HumanosRESUMO
A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defines a new lineage for B. anthracis.
Assuntos
Antraz/epidemiologia , Bacillus anthracis/classificação , Bacillus anthracis/genética , Polimorfismo de Nucleotídeo Único , Antraz/microbiologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , DNA Bacteriano/química , Genótipo , Humanos , Exposição por Inalação , Filogenia , Federação Russa/epidemiologiaRESUMO
In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which "weighs" PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.
Assuntos
Campylobacter/classificação , Campylobacter/genética , Espectrometria de Massas/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Campylobacter/química , Campylobacter coli/classificação , Campylobacter coli/genética , Campylobacter jejuni/classificação , Campylobacter jejuni/genética , Primers do DNA , DNA Bacteriano/análise , Genótipo , Humanos , Especificidade da EspécieRESUMO
We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.
Assuntos
Infecções por Adenoviridae/diagnóstico , Infecções por Adenoviridae/virologia , Adenoviridae/classificação , Adenoviridae/isolamento & purificação , Microbiologia Ambiental , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Adenoviridae/genética , Chlamydiales , Primers do DNA/genética , Processamento Eletrônico de Dados , Humanos , Sensibilidade e Especificidade , Sorotipagem/métodosRESUMO
Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.
Assuntos
Aedes/virologia , Alphavirus/isolamento & purificação , Culex/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Alphavirus/classificação , Alphavirus/genética , Animais , Composição de Bases , Sequência de Bases , Primers do DNA , Humanos , Dados de Sequência Molecular , RNA Viral/análise , RNA Viral/química , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Newly emergent infectious diseases are a global public health problem. The population dense regions of Southeast Asia are the epicenter of many emerging diseases, as evidenced by the outbreak of Nipah, SARS, avian influenza (H5N1), Dengue, and enterovirus 71 in this region in the past decade. Rapid identification, epidemiologic surveillance, and mitigation of transmission are major challenges in ensuring public health safety. Here we describe a powerful new approach for infectious disease surveillance that is based on polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry (ESI-MS) for accurate mass measurements of the PCR products, and base composition signature analysis to identify organisms in a sample. This approach is capable of automated analysis of more than 1,500 PCR reactions a day. It is applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens and will facilitate rapid characterization of known and emerging pathogens.
Assuntos
Doenças Transmissíveis Emergentes/diagnóstico , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Viroses/diagnóstico , Vírus/isolamento & purificação , Controle de Doenças Transmissíveis/métodos , Doenças Transmissíveis Emergentes/microbiologia , Doenças Transmissíveis Emergentes/parasitologia , Doenças Transmissíveis Emergentes/virologia , Humanos , Viroses/virologiaRESUMO
BACKGROUND: Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. METHODS AND PRINCIPAL FINDINGS: Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. CONCLUSION/SIGNIFICANCE: Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.
Assuntos
Vírus da Influenza A/genética , Vigilância da População , Espectrometria de Massas por Ionização por Electrospray/métodos , Genótipo , Vírus da Influenza A/classificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Mitochondrial DNA (mtDNA) mutations cause a large spectrum of clinically important neurodegenerative, neuromuscular, cardiovascular, and endocrine disorders. We describe the novel application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) to the rapid and accurate identification of pathogenic mtDNA variants. METHODS: In a blinded study, we used ESI-FTICR MS to analyze 24 unrelated samples of total cellular DNA containing 12 mtDNA variants and compared the results with those obtained by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and gel electrophoresis. RESULTS: From the 24-sample blinded panel, we correctly identified 12 of the samples as bearing an mtDNA variant and found the remaining 12 samples to have no pathogenic variants. The correlation coefficient between the 2 methods for mtDNA variant detection was 1.0; there were no false positives or false negatives in this sample set. In addition, the ESI-FTICR method identified 4 single-nucleotide polymorphisms (SNP) that had previously been missed by standard PCR-RFLP analysis. CONCLUSIONS: ESI-FTICR MS is a rapid, sensitive, and accurate method for the identification and quantification of mtDNA mutations and SNPs.
Assuntos
DNA Mitocondrial/genética , Análise de Fourier , Genótipo , Humanos , Doenças Mitocondriais/genética , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.