RESUMEN
Extensive use of antibiotics in both public health and animal husbandry has resulted in rapid emergence of antibiotic resistance in almost all human pathogens, including biothreat pathogens. Antibiotic resistance has thus become a major concern for both public health and national security. We developed multiplexed assays for rapid, simultaneous pathogen detection and characterization of ciprofloxacin and doxycycline resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. These assays are SNP-based and use Multiplexed Oligonucleotide Ligation-PCR (MOL-PCR). The MOL-PCR assay chemistry and MOLigo probe design process are presented. A web-based tool - MOLigoDesigner (http://MOLigoDesigner.lanl.gov) was developed to facilitate the probe design. All probes were experimentally validated individually and in multiplexed assays, and minimal sets of multiplexed MOLigo probes were identified for simultaneous pathogen detection and antibiotic resistance characterization.
Asunto(s)
Farmacorresistencia Microbiana/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Animales , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/genética , Bacillus anthracis/patogenicidad , Ciprofloxacina/farmacología , Biología Computacional , ADN Bacteriano/genética , Doxiciclina/farmacología , Francisella tularensis/efectos de los fármacos , Francisella tularensis/genética , Francisella tularensis/patogenicidad , Humanos , Internet , Técnicas de Sonda Molecular , Sondas de Oligonucleótidos/genética , Yersinia pestis/efectos de los fármacos , Yersinia pestis/genética , Yersinia pestis/patogenicidadRESUMEN
We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the ability of this assay to simultaneously detect diverse nucleic acid signatures (e.g., unique sequences, single nucleotide polymorphisms) in a single multiplex reaction. Detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. To demonstrate the utility of our assay, we applied it to the detection of three biothreat agents, B. anthracis, Y. pestis, and F. tularensis. Combined with the ease and robustness of this assay, the results presented here show a strong potential of our assay for use in diagnostics and surveillance.
Asunto(s)
Técnicas de Laboratorio Clínico , Reacción en Cadena de la Ligasa/métodos , Análisis por Micromatrices/métodos , Técnicas de Diagnóstico Molecular/métodos , Bacillus anthracis/genética , Bacillus anthracis/aislamiento & purificación , Francisella tularensis/genética , Francisella tularensis/aislamiento & purificación , Humanos , Microesferas , Yersinia pestis/genética , Yersinia pestis/aislamiento & purificaciónRESUMEN
BACKGROUND: Persistent infection with human papillomavirus type 16 (HPV16) is a primary etiological factor for the development of cervical cancer. Genes involved in antigen processing influence both the repertoire of antigens presented by HPV16-infected cells and the nature of HPV16-specific immune responses. Genetic variation in these genes may affect protein structure and function and, consequently, the ability of an individual to clear HPV infection. METHODS: Thirty-five single-nucleotide polymorphisms (SNPs) in 5 genes (LMP2, TAP1, LMP7, TAP2, and Tapasin) were investigated for association with susceptibility to HPV16-associated cervical cancer. Sequencing of these genes resulted in the discovery of 15 previously unreported SNPs. Microsphere-array flow cytometry-based genotyping was conducted on 787 samples from Hispanic and non-Hispanic white women (241 randomly selected control subjects, 205 HPV16-positive control subjects, and 341 HPV16-positive case subjects with cervical cancer). RESULTS: For 9 SNPs, 8 of which had not previously been reported in the context of cervical cancer, there were statistically significant differences between the genotype distribution in case subjects and that in control subjects. Haplotype analysis of 3 haplotype blocks revealed 3 haplotypes with significant differences in frequency in case-control comparisons. Both HPV16-specific and non-type-specific differences in genotype distribution were seen. CONCLUSIONS: Genes involved in antigen processing for HLA class I presentation may contribute to susceptibility to cervical cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Variación Genética , Antígenos de Histocompatibilidad Clase I/genética , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/genética , Polimorfismo de Nucleótido Simple , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Estudios de Casos y Controles , Cisteína Endopeptidasas/genética , Femenino , Humanos , Proteínas de Transporte de Membrana/genética , Complejos Multienzimáticos/genética , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Valores de ReferenciaRESUMEN
Recently, the phylogenetic placement of the African slender snouted crocodile, Crocodylus cataphractus, has come under scrutiny and herein we address this issue using molecular and morphological techniques. Although it is often recognized as being a "basal" form, morphological studies have traditionally placed C. cataphractus within the genus Crocodylus, while molecular studies have suggested that C. cataphractus is very distinct from other Crocodylus. To address the relationship of this species to its congeners we have sequenced portions of two nuclear genes (C-mos 302bp and ODC 294bp), and two mitochondrial genes (ND6-tRNA(glu)-cytB 347bp and control region 457bp). Analyses of these molecular datasets, both as individual gene sequences and as concatenated sequences, support the hypothesis that C. cataphractus is not a member of Crocodylus or Osteolaemus. Examination of 165 morphological characters supports and strengthens our resurrection of an historic genus, Mecistops (Gray 1844) for cataphractus.
Asunto(s)
Caimanes y Cocodrilos/clasificación , Filogenia , Aldehído Oxidorreductasas/genética , Caimanes y Cocodrilos/anatomía & histología , Caimanes y Cocodrilos/genética , Animales , Núcleo Celular/genética , Citocromos b/genética , ADN Mitocondrial/genética , Genes mos/genética , NADH Deshidrogenasa/genética , Ornitina Descarboxilasa/genéticaRESUMEN
BACKGROUND: Polymorphisms in the TNF-alpha promoter region have recently been shown to be associated with susceptibility to cervical cancer. Some polymorphisms have been reported to influence transcription for this cytokine. Altered local levels in the cervix may influence an individual's immune response, thereby affecting persistence of human papillomavirus (HPV) 16 infection, a primary etiological factor for cervical cancer. METHODS AND RESULTS: The association of 11 TNF-alpha single-nucleotide polymorphisms (SNPs) with susceptibility to HPV16-associated cervical cancer was investigated. Sequencing of the TNF-alpha promoter region confirmed 10 SNPs, and 1 previously unreported SNP (161 bp upstream of the transcriptional start site) was discovered. Microsphere-array flow cytometry-based genotyping was performed on 787 samples from Hispanic and non-Hispanic white women (241 from randomly selected control subjects, 205 from HPV16-positive control subjects, and 341 from HPV16-positive subjects with cervical cancer). The genotype distribution of 3 SNPs (-572, -857, and -863) was significantly different between case subjects and control subjects. Analysis of haplotypes, which were computationally inferred from genotype data, also revealed statistically significant differences in haplotype distribution between case subjects and control subjects. CONCLUSIONS: We report new associations between several TNF-alpha SNPs and susceptibility to cervical cancer that support the involvement of the TNF- alpha promoter region in development of cervical cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Papillomaviridae , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Factor de Necrosis Tumoral alfa/genética , Neoplasias del Cuello Uterino/genética , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Genotipo , Haplotipos , Hispánicos o Latinos , Humanos , Infecciones por Papillomavirus/etnología , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias del Cuello Uterino/etnología , Neoplasias del Cuello Uterino/virología , Población BlancaAsunto(s)
Dermatoglifia del ADN , Personajes , Antropología Forense , Huesos/química , ADN/análisis , ADN/genética , ADN/historia , ADN Mitocondrial/química , ADN Mitocondrial/genética , Historia del Siglo XX , Humanos , Reacción en Cadena de la Polimerasa , Federación de Rusia , Conservación de TejidoRESUMEN
The detection and identification of microbial pathogens are critical challenges in clinical medicine and public health surveillance. Advances in genome analysis technology are providing an unprecedented amount of information about bacterial and viral organisms, and hold great potential for pathogen detection and identification. In this paper, a rational approach to the development and application of nucleic acid signatures is described based on phylogenetically informative sequence features, especially single nucleotide polymorphisms. The computational tools that are available to enable the development of the next generation of microbial molecular signatures for clinical diagnostics and infectious disease surveillance are reviewed and the impact on public health and national security will be discussed.
Asunto(s)
Bacterias/genética , Genética Microbiana/métodos , Técnicas de Diagnóstico Molecular , Ácidos Nucleicos/análisis , Análisis de Secuencia de ADN , Virus/genética , Enfermedades Transmisibles/diagnóstico , Enfermedades Transmisibles/genética , Enfermedades Transmisibles/microbiología , Biología Computacional , Evolución Molecular , Genómica , Humanos , Filogenia , Análisis de Secuencia de ADN/métodosRESUMEN
Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5' end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be 'demultiplexed'. Such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.