RESUMEN
Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis which occurs throughout Africa, Eastern Europe, and Asia and results in an approximately 30% fatality rate. A reverse transcription-PCR assay including a competitive internal control was developed on the basis of the most up-to-date genome information. Biotinylated amplification products were hybridized to DNA macroarrays on the surfaces of polymer supports, and hybridization events were visualized by incubation with a streptavidin-horseradish peroxidase conjugate and the formation of a visible substrate precipitate. Optimal assay conditions for the detection of as few as 6.3 genome copies per reaction were established. Eighteen geographically and historically diverse CCHF virus strains representing all clinically relevant isolates were detected. The feasibility of the assay for clinical diagnosis was validated with acute-phase patient samples from South Africa, Iran, and Pakistan. The assay provides a specific, sensitive, and rapid method for CCHF virus detection without requiring sophisticated equipment. It has usefulness for the clinical diagnosis and surveillance of CCHF infections under limited laboratory conditions in developing countries or in field situations.
Asunto(s)
Virus de la Fiebre Hemorrágica de Crimea-Congo/aislamiento & purificación , Fiebre Hemorrágica de Crimea/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Humanos , Irán , Sondas de Oligonucleótidos/genética , Pakistán , Sensibilidad y Especificidad , SudáfricaRESUMEN
KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.
Asunto(s)
Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , ADN/análisis , Pruebas Genéticas/métodos , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , ADN/genética , Análisis Mutacional de ADN/economía , Análisis Mutacional de ADN/métodos , Formaldehído , Pruebas Genéticas/economía , Genotipo , Humanos , Desnaturalización de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/economía , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adhesión en Parafina , Proteínas Proto-Oncogénicas p21(ras) , Fijación del TejidoRESUMEN
A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.
Asunto(s)
Coronavirus/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Técnicas de Tipificación Bacteriana , Coronavirus/genética , Coronavirus/aislamiento & purificación , Coronavirus Humano 229E/clasificación , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/aislamiento & purificación , Coronavirus Humano OC43/clasificación , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/aislamiento & purificación , Humanos , ARN Viral/análisis , ARN Viral/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/clasificación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Especificidad de la EspecieRESUMEN
BACKGROUND: A new, fast 'low cost and density' DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona. METHODS: LCD chips are pre-structured polymer supports using a non-fluorescent detection principle based on the precipitation of a clearly visible dark substrate. One LCD chip consists of eight identical microarrays, designed to detect mutations within the 90 bp rpoB region, codon 315 in the katG gene and the mabA-inhA regulatory region. A total of 22 strains with a katG 315 mutation, 19 strains with alterations in the mabA-inhA regulatory region and 16 strains with mutations in the rpoB region, characterized previously, were studied. RESULTS: The identification of S315T and S315N mutations using the LCD was 100% concordant with the sequencing data. A strain with the S315R mutation, which is not tiled on the LCD array, was detected by the absence of hybridization using the wild-type probe. Of 19 strains with low-level isoniazid resistance related to the mabA-inhA regulatory region, 18 were identified correctly. The detection of mutations in the rpoB region was 93.8% concordant with the sequencing data. One mabA-inhA and rpoB mutated strain showed a cross-hybridization. CONCLUSIONS: The LCD array protocol takes 45 min (15 min 'hands-on' time) after prior PCR amplification. Only minimal laboratory equipment is required. LCD arrays provide a rapid and economical method to characterize mutations in codon 315 of the katG gene, in the mabA-inhA regulatory region and in the rpoB gene.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Genes Bacterianos , Mutación , Mycobacterium tuberculosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Bacterianas/genética , Catalasa/genética , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN , Humanos , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Rifampin/farmacología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation in vitro. Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of approximately 184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethyl-benzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation in vivo. These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders.