RESUMO
We investigated a number of PCR-based strategies for sub-typing isolates of Mycobacterium bovis. A panel of 15 PCR primers, designed from random sequences, insertion sequences and repetitive elements, were analyzed for their ability to allow differentiation between eight M. bovis isolates. PCR products were analyzed using agarose gel electrophoresis and polyacrylamide gel electrophoresis. Random primer based PCR, conducted with a variety of primers did not differentiate between isolates of M. bovis. Successful differentiation between isolates was achieved by the amplification of fragments located between DNA repetitive elements and insertion sequences of the M. bovis genome. PCR primers designed from the major polymorphic tandem repeat (MPTR) region and from insertion sequences IS6110 and IS986 allowed differentiation between isolates of M. bovis. This study is presented as a first step towards the development of PCR-based methods to differentiate between isolates.
Assuntos
Impressões Digitais de DNA/métodos , Mycobacterium bovis/classificação , Mycobacterium bovis/isolamento & purificação , Animais , Sequência de Bases , Bovinos , Primers do DNA , DNA Bacteriano/isolamento & purificação , Cervos , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Marcadores Genéticos , Humanos , Mycobacterium bovis/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo GenéticoRESUMO
The polymerase chain reaction plays a central role in many detection assays and methods to improve the sensitivity and specificity of these detection systems are constantly being explored. In this study we investigated the use of an automated laser fluorescent system (ALF) in the context of DNA-based diagnostics for pathogenic bacteria. PCR products were generated using species-specific primer sets, one of which was labelled with a 5' fluorescein. PCR products with a fluorescent label were detected on line with an ALF DNA sequencer and the sensitivity of detection was found to be comparable to that for DNA probe hybridization with a radioactive probe. The technology was successfully applied to the detection of Mycobacterium tuberculosis supplemented into sputum samples and to the detection of listeria in paraffin-embedded tissue samples.
Assuntos
Corantes Fluorescentes , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Humanos , Listeria/isolamento & purificação , Dados de Sequência Molecular , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologiaRESUMO
OBJECTIVES: To identify the rate of baby walker use, parental attitudes, and associated injuries. DESIGN: Parents of babies attending clinics for developmental assessment were surveyed by self administered questionnaire about their use, attitudes, and history of injuries associated with walkers. SETTING: Dublin, Ireland. SUBJECTS: Parents of 158 babies. RESULTS: Fifty five per cent of the sample used a walker. The main reasons for doing so included babies' enjoyment of them and the fact that the walker was used for an older sibling. Although none of the users listed safety concerns as a reason to stop using the walker, non-users (45%) did so; 12.5% of the users had at least one walker related injury. CONCLUSIONS: Parents of babies who use a walker perceive them as beneficial. However these babies are placed at unnecessary risk. It behoves all health professionals and child carers to alert parents to these dangers and the sale of walkers should be reviewed.
