RESUMO
Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.
Assuntos
Eletroquímica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sistema Respiratório/microbiologia , Sistema Respiratório/virologia , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Bordetella pertussis/genética , Bordetella pertussis/isolamento & purificação , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/isolamento & purificação , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/isolamento & purificação , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Viral/química , DNA Viral/genética , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/isolamento & purificação , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 1 Humana/isolamento & purificação , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/isolamento & purificação , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/isolamento & purificação , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação , Viroses/diagnóstico , Viroses/virologiaRESUMO
In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.
Assuntos
Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Neuraminidase/genética , Vírus da Influenza A/enzimologia , Vírus da Influenza A/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Semicondutores , Análise de Sequência de DNARESUMO
The comparative-genomic sequencing of two Mycobacterium tuberculosis strains enabled us to identify single nucleotide polymorphism (SNP) markers for studies of evolution, pathogenesis, and epidemiology in clinical M. tuberculosis. Phylogenetic analysis using these "comparative-genome markers" (CGMs) produced a highly unusual phylogeny with a complete absence of secondary branches. To investigate CGM-based phylogenies, we devised computer models to simulate sequence evolution and calculate new phylogenies based on an SNP format. We found that CGMs represent a distinct class of phylogenetic markers that depend critically on the genetic distances between compared "reference strains." Properly distanced reference strains generate CGMs that accurately depict evolutionary relationships, distorted only by branch collapse. Improperly distanced reference strains generate CGMs that distort and reroot outgroups. Applying this understanding to the CGM-based phylogeny of M. tuberculosis, we found evidence to suggest that this species is highly clonal without detectable lateral gene exchange. We noted indications of evolutionary bottlenecks, including one at the level of the PHRI "C" strain previously associated with particular virulence characteristics. Our evidence also suggests that loss of IS6110 to fewer than seven elements per genome is uncommon. Finally, we present population-based evidence that KasA, an important component of mycolic acid biosynthesis, develops G312S polymorphisms under selective pressure.
