RESUMO
A rapid and readily available DNA probe kit was developed for the detection of Salmonella spp. This kit utilized the colorimetric DNA/rRNA sandwich hybridization method in microtiter wells. Within 3 hr Salmonella spp. in selective enrichment broth cultures were detected by the DNA probe kit. The kit effectively identified all of 187 strains of Salmonella tested and yielded no false-positive reactions in the examination of 674 pure cultures of non-salmonellae. The DNA probe kit could detect 10(5) cfu/ml in pure culture. A total of 379 naturally contaminated samples (raw chicken meat, liquid egg, animal feeds, poultry feces and frozen foods) were tested, both by the standard culture method and the DNA probe kit. The 169 of these samples were culture positive and 210 were culture negative. The sensitivity of the DNA probe kit was 98.2% (166/169) and the specificity was 99.5% (209/210). These results show that the DNA probe kit is a useful tool to examine a large number of various samples for contamination by Salmonella spp. in food and livestock industry.
Assuntos
Hibridização de Ácido Nucleico/métodos , Saúde Pública , Intoxicação Alimentar por Salmonella/prevenção & controle , Salmonella typhimurium/isolamento & purificação , Ração Animal/microbiologia , Animais , Galinhas , Contagem de Colônia Microbiana , Colorimetria , DNA Bacteriano/química , Ovos/microbiologia , Fezes/microbiologia , Alimentos Congelados/microbiologia , Humanos , Carne/microbiologia , RNA Ribossômico/química , Kit de Reagentes para Diagnóstico , Salmonella typhimurium/genética , Sensibilidade e Especificidade , SuínosRESUMO
The DNA sequence of the gene encoding the early and specific 46-kDa surface antigen (P46) of Mycoplasma hyopneumoniae has been determined. The P46 gene, encoding a putative lipoprotein, contained three TGA codons and a single CGG codon in a 1,257-bp open reading frame. Edman degradation of peptide fragments showed that at least one TGA codon encodes tryptophan and that the CGG codon, which has been reported to be nonsense or unassigned in other mycoplasmas, is used for arginine in M. hyopneumoniae.
Assuntos
Antígenos de Bactérias/genética , Arginina/genética , Proteínas de Bactérias/genética , Códon , Genes Bacterianos , Mycoplasma/imunologia , Sequência de Aminoácidos , Antígenos de Superfície/genética , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Peso Molecular , Mycoplasma/genéticaRESUMO
The 46-kDa surface antigen (P46) is the early and species-specific immunogenic protein of Mycoplasma hyopneumoniae. Three TGA codons encoding tryptophan in the P46 gene were replaced with TGG by an in vitro mutagenesis technique. The mutated P46 gene was expressed in Escherichia coli by using the chelating peptide tag system. The purified recombinant P46 was successfully used in an enzyme-linked immunosorbent assay for detection of antibodies against M. hyopneumoniae in swine serum. It did not cross-react with sera from swine infected with Mycoplasma flocculate, Mycoplasma hyorhinis, or Mycoplasma hyosynoviae. With this method, mycoplasmal pneumonia of swine was detectable within 2 weeks after infection.
Assuntos
Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Mycoplasma/imunologia , Pneumonia Suína Micoplasmática/veterinária , Doenças dos Suínos/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/análise , Proteínas de Bactérias/genética , Sequência de Bases , Códon , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Mycoplasma/genética , Pneumonia Suína Micoplasmática/diagnóstico , Pneumonia Suína Micoplasmática/genética , Pneumonia Suína Micoplasmática/imunologia , Mutação Puntual , Proteínas Recombinantes/imunologia , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/imunologiaRESUMO
Bordetella bronchiseptica 16S ribosomal RNA (rRNA) gene was cloned and identified. On the basis of information from computer-assisted sequence comparison of the B. bronchiseptica 16S RRNA sequences with that of other bacterial species, we constructed B. bronchiseptica-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Specificity of these 32P-labeled oligo-nucleotide probes was tested in a RNA/DNA hybridization with B. bronchiseptica strains and other bacterial strains. Probe BB4 was more specific than three other oligonucleotide probes. This probe BB4 was sensitive enough to be able to detect 10(4) bacterial cells.
Assuntos
Bordetella bronchiseptica/isolamento & purificação , Sondas de Oligonucleotídeos/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sequência de Bases , Bordetella bronchiseptica/genética , Dados de Sequência Molecular , Sensibilidade e EspecificidadeRESUMO
A system that uses rRNA-oligodeoxynucleotide hybridization was developed for the detection of Mycoplasma hyopneumoniae. Synthetic oligonucleotide MHP1 was hybridized specifically with M. hyopneumoniae. Furthermore, the detection of M. hyopneumoniae in clinical samples, such as bronchoalveolar lavage fluid and lung lesions from experimentally infected pigs, was evaluated by this assay. The evidence obtained from the assay indicated that the system can be used to efficiently diagnose mycoplasmal pneumonia of swine. Additionally, a nonradioisotopic system with chemiluminescence detection was tested. This system was 10-fold less sensitive than a test that used radioisotopes.
