RESUMEN
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.
Asunto(s)
Infección Hospitalaria/prevención & control , Genética Microbiana/métodos , Epidemiología Molecular/métodos , Vigilancia de la Población/métodos , Infecciones por Acinetobacter/epidemiología , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/genética , Acinetobacter baumannii/aislamiento & purificación , Infección Hospitalaria/diagnóstico , Infección Hospitalaria/microbiología , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Brotes de Enfermedades , Genes Bacterianos , Genotipo , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Ionización de Electrospray/métodos , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Streptococcus pyogenes/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Campylobacter/clasificación , Campylobacter/genética , Espectrometría de Masas/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Composición de Base , Campylobacter/química , Campylobacter coli/clasificación , Campylobacter coli/genética , Campylobacter jejuni/clasificación , Campylobacter jejuni/genética , Cartilla de ADN , ADN Bacteriano/análisis , Genotipo , Humanos , Especificidad de la EspecieRESUMEN
In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.
Asunto(s)
Composición de Base , ADN Mitocondrial/química , Ciencias Forenses/métodos , Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Ionización de Electrospray/métodos , Automatización , Secuencia de Bases , ADN/sangre , Enzimas de Restricción del ADN/metabolismo , Bases de Datos de Ácidos Nucleicos , Humanos , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.
Asunto(s)
Bacterias/genética , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/microbiología , Vigilancia de Guardia , Factores de Virulencia/genética , Composición de Base , California/epidemiología , Secuencia Conservada/genética , Cartilla de ADN , Técnicas Genéticas , Genotipo , Humanos , Personal Militar , Especificidad de la Especie , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was approximate, equals1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.
Asunto(s)
Infecciones por Coronaviridae/virología , Coronaviridae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Secuencia de Bases , Enfermedades Transmisibles Emergentes , Infecciones por Coronaviridae/epidemiología , Humanos , Vigilancia de la Población , ARN Viral , Sensibilidad y EspecificidadRESUMEN
Fourier transform ion cyclotron resonance (FTICR) mass spectrometry represents a unique platform with which to study nucleic acids and non-covalent complexes containing nucleic acids moieties. In particular, systems in which very high mass measurement accuracy is required, very complex mixtures are to be analyzed, or very limited amounts of sample are available may be uniquely suited to interrogation by FTICR mass spectrometry. Although the FTICR platform is now broadly deployed as an integral component of many high-end proteomics-based research efforts, momentum is still building for the application of the platform towards nucleic acid-based analyses. In this work, we review fundamental aspects of nucleic acid analysis by FTICR, focusing primarily on the analysis of DNA oligonucleotides but also describing applications related to the characterization of RNA constructs. The goal of this review article is to give the reader a sense of the breadth and scope of the status quo of FTICR analysis of nucleic acids and to summarize a few recently published reports in which researchers have exploited the performance attributes of FTICR to characterize nucleic acids in support of basic and applied research disciplines including genotyping, drug discovery, and forensic analyses.
Asunto(s)
Ciclotrones , Espectrometría de Masas/métodos , Ácidos Nucleicos/análisis , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.
Asunto(s)
Bacillus anthracis/genética , Polimorfismo de Nucleótido Simple/genética , Espectrometría de Masa por Ionización de Electrospray/métodos , Alelos , Bacillus anthracis/química , Bioterrorismo , Análisis de Fourier , Marcadores Genéticos , Genotipo , Reacción en Cadena de la Polimerasa , Secuencias Repetidas en TándemRESUMEN
In this work we present variations on in-hexapole infrared multiphoton dissociation (IRMPD) for the characterization of modified oligonucleotides using an ESI-FTICR spectrometer. We demonstrate that IRMPD in the external ion reservoir provides a comprehensive series of fragments allowing thorough characterization of a wide range of oligonucleotides containing alternative backbones and 2' substitutions. An alternative pulse sequence is presented that allows alternating MS and IRMPD MS/MS spectra to be acquired on a chromatographic timescale without loss in ionization duty cycle. Ions are excited to a larger cyclotron radius such that they "dodge" the IR laser beam that travels through the center of the trapped ion cell and impinges on the external ion reservoir creating IRMPD fragments that will be detected in the next scan. An alternative approach for directing IR radiation into the external ion reservoir using a hollow fiber waveguide as a photon conduit is presented. This approach offers a simple and robust alternative to the previously utilized on-axis scheme and may allow effective implementation with lower power lasers owing to the inherent increase in power density achieved by focusing the nascent laser beam into the hollow fiber waveguide.
Asunto(s)
Oligonucleótidos/química , Cromatografía Líquida de Alta Presión , Ciclotrones , Análisis de Fourier , Rayos Infrarrojos , Rayos Láser , Espectrometría de Masas , FotonesRESUMEN
A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.6-microm IR laser beam, initially 3.5 mm in diameter, is focused into the vacuum-sealed HFWG. The small internal diameter and the high transfer efficiency of the waveguide allow IR radiation of high power density to be employed for IRMPD. In studies performed on a quadrupole ion trap, a 500-microm-i.d. waveguide was used as a medium to transmit IR radiation directly through a 700-microm orifice in the ring electrode. Efficient IRMPD of both a 12-mer oligonucleotide and the protein melittin were performed at laser powers of 0.5 and 3.2 W, respectively.