Development of a Colorimetric, Second Generation Nucleic Acid Hybridization Method For Detection of Salmonella in Foods and a Comparison With Conventional Culture Procedure

Wilson, Stephen G.; Chan, S. H.; Deroo, M; Vera-García, M.E.; Johnson, Andrew D.; Lane, David; Halbert, Donald N.

doi:10.1111/j.1365-2621.1990.tb03944.x

Cited by 25 publications

(17 citation statements)

References 22 publications

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“…were compared to standard culture methods by naturally contaminated samples [5,10,13,21]. In this study, a total of 379 naturally contaminated samples were tested.…”

Section: Discussionmentioning

confidence: 99%

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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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“…were compared to standard culture methods by naturally contaminated samples [5,10,13,21]. In this study, a total of 379 naturally contaminated samples were tested.…”

Section: Discussionmentioning

confidence: 99%

“…in food samples [4,21]. This kit used the dipstick format which is one of the solid-phase sandwich hybridization format [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2000

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“…In addition, to further confirm the presence of non-Salmonella DNAs on the filters, the authors have also carried out a hybridization study for the DNAs from the non-Salmonella isolates using a non-specific probe which is universal to all the non-Salmonella isolates. The probe used was an oligonucleotide probe derived from the conserved region of the 16s rRNA gene, for example, the probe of P3mod, which had been proved to be universal to eubacteria 16s rRNA genes (Wilson et al 1990a). Its sequence was 5'-ATTAGATACCCTGGTAGTCC-3'.…”

Section: Sequencing the V3 To V6 Region Of Salm Enterltldls 16s Rrnamentioning

confidence: 99%

“…In such a case, however, the enrichment step is still important since it would assure the positive detection of target organisms, especially when the target cell numbers are below lo2 cells ml-of the sample. The media generally used for the enrichment of Salmonella in food samples are lactose broth (LB), tetrathionate (TT) and Gram-negative (GN) broth (Flowers et al 1987;Emswiler-Rose et al 1987;Curiale et al 1990;Wilson et al 1990b). For example, when a commercial Gene-Trak system was used for the detection of Salmonella in food samples, enrichment of target cells using LB followed by TT broth and G N broth was generally required prior to the hybridization steps (Flowers et al 1987;Wilson et al 1990b).…”

Section: Sequencing the V3 To V6 Region Of Salm Enterltldls 16s Rrnamentioning

confidence: 99%

Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods

Lin

Tsen

1995

Journal of Applied Bacteriology

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DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non-Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella.

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“…Recent work on nonisotopic labels is promising and should lead to a broad implementation of 206 SEAFOODS this technique (Gopo et aI., 1988;Romick et aI., 1989;Wilson et al, 1990;Gicquelais et al, 1991;Peterkin et al, 1991). Because of the sample dilution that occurs during blending and because of limitations on the amount of liquid that can be spread on an agar plate, the colony hybridization test has a practical lower limit of detection of about 20-100 cells per gram.…”

Section: Gene Probesmentioning

confidence: 99%

Seafoods: Chemistry, Processing Technology and Quality

Shahidi¹,

Botta²

1994

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Development of a Colorimetric, Second Generation Nucleic Acid Hybridization Method For Detection of Salmonella in Foods and a Comparison With Conventional Culture Procedure

Cited by 25 publications

References 22 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 and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods

Seafoods: Chemistry, Processing Technology and Quality

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