Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences

Taneda, Atsuyoshi; Futo, Satoshi; Mitsuse, Shizuo; Seto, Yasuhiro; Okada, Mizue; Sakano, Tetsuya

doi:10.1016/0378-1135(94)90061-2

Veterinary Microbiology

1994

DOI: 10.1016/0378-1135(94)90061-2

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Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences

Atsuyoshi Taneda

Satoshi Futo

Shizuo Mitsuse

et al.

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Cited by 3 publications

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“…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].…”

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Detection of Salmonella by Using the Colorimetric DNA/rRNA Sandwich Hybridization in Microtiter Wells.

et al. 2000

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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 ...

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Detection of Salmonella by Using the Colorimetric DNA/rRNA Sandwich Hybridization in Microtiter Wells.

et al. 2000

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Development of a Rapid Salmonella Detection Kit by the Colorimetric DNA/RNA Hybridization Method in Microtiter Wells.

Namimatsu

Tsuna

Imai

et al. 1998

Jpn.J.Food Microbiol.

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Namimatsu

Ogawa

Iida

et al. 2001

Jpn.J.Food Microbiol.

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Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences

Cited by 3 publications

References 25 publications

Detection of Salmonella by Using the Colorimetric DNA/rRNA Sandwich Hybridization in Microtiter Wells.

Detection of Salmonella by Using the Colorimetric DNA/rRNA Sandwich Hybridization in Microtiter Wells.

Development of a Rapid Salmonella Detection Kit by the Colorimetric DNA/RNA Hybridization Method in Microtiter Wells.

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