Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure

Abstract

To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a SalmoneUla-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonela organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100lo compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.

Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identifySalmonella typhimuriumin food

“…from food. The primers are different from those suggested in previous studies by Jitrapakdee et al (1995), Stone et al (1994) and Rahn et al (1992). The differences consist in the sequences and in the annealing sites on the nucleotide sequence of the invA gene.…”

“…The method potentially allows amplification of the target DNA from as few as one copy (Zhu et al 1996). Several PCR methods of detecting Salmonella have been published utilizing a specific gene sequence for targeting (Way et al 1993;Stone et al 1994;Lin and Tsen 1996;Lampel et al 1996;Zhu et al 1996;Kwang et al 1996).…”

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

“…The sensitivity of detection of V. cholerae organisms in prepared environmental samples was, in some instances, lower than that in pure cultures. Others have reported similar results of sensitivities with clinical and environmental samples (Hermans et al 1990;Shawar et al 1993;Stone et al 1994). It should be noted, however, that the bacteria used to seed the samples were freshly grown.…”

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

“…Several PCR assays for detection of Salmonella have been developed, and several different target DNAs for amplification have been applied. Polymerase chain reaction assays which enable the detection of Salmonella in different sources, such as human or animal faeces (Widjojoatmodjo et al 1992;Mahon and Lax 1993;Cohen et al 1994aCohen et al , 1994bCohen et al , 1995Kongmuang et al 1994;Stone et al 1994;Haedicke et al 1996), various food samples (Cano et al 1993;Fluit et al 1993;Chen et al 1997), fish meat (Iida et al 1993;Lin and Tsen 1996), various meat products (Soumet et al 1994(Soumet et al , 1997Aabo et al 1995;Jitrapakdee et al 1995;Kwang et al 1996;Lin and Tsen 1996), chicken skin or organs (Mahon et al 1994;Tuchili et al 1995), eggs (Burkhalter et al 1995;Bäumler et al 1997), dairy products (Chevrier et al 1995;Cohen et al 1996), oysters (Jones et al 1993Bej et al 1994;Brasher et al 1998), feed (Cohen et al 1996), soil (Way et al 1993) and environmental water samples (Bej et al 1990b(Bej et al , 1991bWay et al 1993) have been described. The few assays described for the detection of Salmonella in water have been applied to water samples with low turbidity, such as autoclaved tap water and well water.…”