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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 falsepositive 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.-KEY WORDS: DNA probe kit, hybridization, rapid detection kit, rRNA, Salmonella. Salmonella spp. is one of the most important pathogenic bacteria for humans and animals [3]. Therefore, it is very important to detect for Salmonella spp. from diarrheal stools of salmonellosis in the humans and the animals, foods, feeds and environmental samples. For increase of detection rate of Salmonella spp. from these samples, the culture methods and the selective media were modified and developed [7,14,17]. But the culture methods for Salmonella spp. can take 5-6 days until complete judgement [19].In the past years, DNA probes hybridizing to the variable region of bacterial ribosomal RNA (rRNA) have been developed to identify some bacterial species [6,11,20]. For the specific detection of Salmonella spp., we developed the visual colorimetric DNA/rRNA sandwich hybridization system derived from the 23S rRNA gene of Salmonella Typhimurium in microtiter wells [15]. This system can reduce the period of detecting Salmonella spp. from samples by up to 2 days.In this paper, this hybridization system was examined for the specificity and sensitivity using pure culture samples and was compared with standard culture method for the detection of Salmonella spp. in many artificially and naturally contaminated samples. MATERIALS AND METHODS T h e v i s ua l c ol o r i m e t r i c D N A/ r RN A s a nd w i c h hybridization system assay:Selective enrichment cultures were probed according to the manufacturer's instructions [15]. Briefly, test samples were exposed to strong alkaline conditions (0.3N NaOH) to release rRNA from bacterial cells. The hybridization buffer containing detector DNA probes and the lysate were placed in microtiter plate wells where capturing DNA for rRNA of salmonella were fixed. Following incubation for 1 hr at 37°C, wells were washed. The solution containing anti-digoxigenin antibodies coupled to the enzyme horseradish peroxidase were added to the wells. Following ...

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.There has been increasing interest in the phylogenetics of mycoplasmas. A small AϩT-rich genome and a specific codon usage, which diverges from the universal genetic code, are unique features of these organisms. In several mycoplasma species, TGA is translated as tryptophan rather than a translational stop (3,19). Also, it was suggested that CGG, an arginine codon in the universal code, is an unassigned or nonsense codon in Mycoplasma capricolum (10, 11) but not in Mycoplasma pneumoniae (3,20). It is thus an ambiguous point in the genetic code of mycoplasmas.Mycoplasma hyopneumoniae is the pathogenic agent of mycoplasmal pneumonia of swine. The 46-kDa antigen (P46) of M. hyopneumoniae is one of the species-specific surface proteins. Recently, the enzyme-linked immunosorbent assay double sandwich method, using a monoclonal antibody which recognizes P46 of M. hyopneumoniae, was successfully applied to the diagnosis of mycoplasmal pneumonia of swine (6, 7). In this report, we describe cloning and the nucleotide sequence of the gene encoding P46 of M. hyopneumoniae and the properties of the gene. The data showed that at least one TGA codon encodes tryptophan and that a CGG codon is used for arginine in M. hyopneumoniae. This is different from M. capricolum, in which the CGG codon is nonsense or unassigned.Cloning of the P46 gene was accomplished by the following experiments. A gt11 genomic library of M. hyopneumoniae ATCC 25934 (strain J) was constructed and immunoscreened with swine hyperimmune serum against the same strain (21). Approximately 500 clones were immunologically reactive among 2 ϫ 10 7 plaques. Subsequently, specific antibodies against recombinant clones were prepared from the hyperimmune serum by epitope selection (18). Each selected antibody was used to probe the immunoblots of M. hyopneumoniae whole-cell proteins for identification of the recombinant products. As a result, two clones which encode almost the same region of P46, NRK02 and NRK03, were obtained.On the basis of the results of genomic Southern analysis with the DNA fragment inserted in NRK02 as a probe, it was suggested that a 5.0-kb HindIII fragment contained the fulllength P46 gene (data not shown). Therefore, a HindIII fragment library was constructed with pUC119 (17). A plasmid clone containing the complete P46 gene, designated pURR126, was obtained by colony hybridization. The sequence of the P46 gene in the HindIII insert was determined by the dideoxy-mediated chain-termination method (Fig. 1) (12).The sequencing result showed that there i...

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