ABSTRACT
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.
Subject(s)
Nucleic Acid Hybridization/methods , Public Health , Salmonella Food Poisoning/prevention & control , Salmonella typhimurium/isolation & purification , Animal Feed/microbiology , Animals , Chickens , Colony Count, Microbial , Colorimetry , DNA, Bacterial/chemistry , Eggs/microbiology , Feces/microbiology , Frozen Foods/microbiology , Humans , Meat/microbiology , RNA, Ribosomal/chemistry , Reagent Kits, Diagnostic , Salmonella typhimurium/genetics , Sensitivity and Specificity , SwineABSTRACT
The roles of tumor necrosis factor (TNF)-alpha and the facilitative glucose transporter (GLUT) 4 on the induction of insulin resistance in peripheral tissues of cancer patients was examined by quantitative competitive PCR on biopsies of abdominal rectal muscle from patients with gastrointestinal cancer. The degree of insulin resistance in these patients was measured by the euglycemic hyperinsulinemic glucose clamp using a high physiologic insulin concentration (100 microU/ml). Quantitative competitive PCR was carried out using DNA competitors constructed by deleting 20-30 bp between the two primer annealing sites. Decreased glucose uptake (M value) in peripheral tissues was accompanied by a significantly increased TNF-alpha mRNA in skeletal muscle (r=0.867, p=0.0025). GLUT4 mRNA, however, was positively correlated with M values (r=0.739, p=0.015). The amounts of mRNAs for TNF-alpha and GLUT4 in skeletal muscle were not correlated. Serum TNF-alpha concentrations remained below the limit of detection. These findings suggest that the insulin resistance in peripheral tissues of cancer patients is in part due to the induction TNF-alpha mRNA and the down regulation of GLUT4 mRNA in peripheral tissues.
Subject(s)
Gastrointestinal Neoplasms/metabolism , Insulin Resistance , Muscle Proteins , Muscle, Skeletal/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Female , Glucose/metabolism , Glucose Clamp Technique , Glucose Transporter Type 4 , Humans , Insulin/pharmacology , Male , Middle Aged , Monosaccharide Transport ProteinsABSTRACT
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.
Subject(s)
Antigens, Bacterial/genetics , Arginine/genetics , Bacterial Proteins/genetics , Codon , Genes, Bacterial , Mycoplasma/immunology , Amino Acid Sequence , Antigens, Surface/genetics , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Molecular Weight , Mycoplasma/geneticsABSTRACT
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.
Subject(s)
Bordetella bronchiseptica/isolation & purification , Oligonucleotide Probes/genetics , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Base Sequence , Bordetella bronchiseptica/genetics , Molecular Sequence Data , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Mycoplasma/genetics , RNA, Bacterial/genetics , RNA, Ribosomal/genetics , Animals , Base Sequence , Enzyme-Linked Immunosorbent Assay/methods , Molecular Sequence Data , Mycoplasma/isolation & purification , Mycoplasma Infections/microbiology , Nucleic Acid Hybridization , Oligonucleotide Probes/chemical synthesis , Sensitivity and Specificity , SwineABSTRACT
Tumor-promoting phorbol esters including 12-O-tetradecanoylphorbol-13-acetate (TPA), specific inhibitors for erythroid differentiation in mouse Friend cells, induce a newly identified nuclear protein with a molecular weight of 54,000 (p54) in Friend cells. Phorbol, an analogue of TPA with neither tumor-promoting nor differentiation-inhibitory activity, did not induce p54. In a variant cell line of Friend cells which exhibits resistance to TPA in erythroid differentiation, p54 was not induced by TPA. The induction of p54 by TPA was counteracted by erythroid-inducing agents including dimethyl sulfoxide, hexamethylenebisacetamide, and actinomycin D. Throughout these experiments, we have observed an inverse relationship between p54 induction and cellular potential for erythroid differentiation.
