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Development of a qPCR for the detection and quantification of Salmonella spp. in sheep feces and tissues.

ABSTRACT: Salmonella spp. are common causes of disease in intensive livestock production systems, and contamination of foodstuffs is of significant concern for public health. Therefore, the identification and quantification of Salmonella spp. is important for monitoring the level of fecal shedding or tissue colonization in infected animals and animal products. We developed and evaluated a quantitative PCR (qPCR) method on spiked sheep tissue and fecal samples for the detection and quantification of Salmonella spp. Without the use of a pre-enrichment step, the qPCR limit of detection (LOD) results for sheep fecal (4 × 10⁴-6 × 10³ cfu/g) and tissue (4 × 10⁵-4 × 10³ cfu/g) samples were not adequate for detection purposes. With the inclusion of a 6-h pre-enrichment step in buffered peptone water (BPW), the LOD was 9 cfu/g (2.57 × 10¹ copies/g) in sheep feces, and 5.4 cfu/g (3.22 copies/g) sheep tissue. Comparison of the 6-h BPW qPCR method with a 24-h mannitol-selenite-cystine broth enrichment culture method using spiked samples revealed a sensitivity of 91% and 92%, respectively, and a specificity of 100% for both methods. The correlation was significant between the quantity (copies/mL) of Salmonella spp. in BPW at 6 h and at 0 h, allowing semiquantitative analysis. Our results demonstrate that, following inclusion of a 6-h pre-enrichment step in BPW, qPCR is semiquantitative with improved LODs of Salmonella spp. in sheep fecal and tissue samples.

SUBMITTER: Parker AM

PROVIDER: S-EPMC7649550 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Development of a qPCR for the detection and quantification of Salmonella spp. in sheep feces and tissues.

Parker Alysia M AM Mohler Virginia L VL Gunn Alison A AA House John K JK

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20200828 6

Salmonella spp. are common causes of disease in intensive livestock production systems, and contamination of foodstuffs is of significant concern for public health. Therefore, the identification and quantification of Salmonella spp. is important for monitoring the level of fecal shedding or tissue colonization in infected animals and animal products. We developed and evaluated a quantitative PCR (qPCR) method on spiked sheep tissue and fecal samples for the detection and quantifica ...[more]

PMID: 32856555

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Project description:BackgroundAlthough a variety of methodologies are available for detection of Salmonella, sensitive, specific, and efficient methods are urgently needed for differentiation of live Salmonella cells from dead cells in food and environmental samples. Propidium monoazide (PMA) can preferentially penetrate the compromised membranes of dead cells and inhibit their DNA amplification, however, such inhibition has been reported to be incomplete by some studies. In the present study, we report an efficient qPCR assay targeting a conserved region of the invA gene of Salmonella in conjunction with PMA treatment for detection of DNA from live Salmonella cells in food samples.ResultsWe investigated the relationship between amplicon length and inhibitory effect of PMA treatment to prevent DNA amplification from dead cells while allowing for DNA amplification from live cells, and found that the two factors are well correlated with each other. An amplicon that is 130 bp in length was determined to be optimal for PMA treatment and was selected for further PMA-qPCR assay development. A PMA-qPCR assay was established by utilizing this amplicon and adopting a modified PMA-treatment procedure. The PMA-qPCR assay provided excellent inhibition of DNA amplification from dead cells (a 17-CT-value, or 128,000-fold reduction) while only a slight DNA amplification difference (0.5 CT value) was noted between the PMA-treated and untreated live cells. This assay has been validated through stringent inclusivity and exclusivity studies using a large number of (n = 167) Salmonella, including all strains of SARA and SARB collections, and non-Salmonella strains (n = 36). This PMA-qPCR assay is capable of detecting live Salmonella cells in live/dead cell mixtures, or 30 CFU/g live Salmonella cells from enriched spiked spinach samples as early as 4 h.ConclusionsA 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells by PCR. This PMA-qPCR assay provides a sensitive, specific, and efficient method for detecting live Salmonella cells in foods and environmental samples and may have an impact on the accurate microbiological monitoring of Salmonella in foods and environment samples.

Project description:Salmonella is a common and important pathogen for both human and animals. All Salmonella except Salmonella pullorum and Salmonella gallinarum have flagellum. Flagellin (FliC) is the main subunit protein forming the bacterial filament, which is present in large amounts on the surface of all flagellated Salmonella. After bioinformatics analysis, the most highly conserved region (locates position from 1 to 102 amino acid residue of FliC, we named it as FliC') was selected, and corresponding recombinant FliC' (rFliC') protein was tailored as an immunogen to generate monoclonal antibodies (MAbs) against Salmonella flagellin. BALB/c mice were immunized with the purified recombinant protein rFliC', which were prepared by prokaryotic expression system pET22b (+) expressing FliC'. After fusion of spleen cells from the immunized mice and SP2/0 cells, three hybridoma cells (1D6, 2G6 and 3E2) producing MAbs against targeted flagellate Salmonella FliC' were generated and screened. The ability of MAb 3E2 to recognize and bind to Salmonella flagella was demonstrated by immunogold electron microscopy (IEM) method. Western blot (WB) analysis demonstrated that MAb 3E2 could specifically recognize flagellated Salmonella strains. Moreover, MAb 3E2 has a direct agglutination activity against Salmonella strains with visible agglutination reaction. To further verify this agglutination activity, a total of 52 flagellated Salmonella strains (23 serovars), 8 non-flagellate Salmonella strains (2 serovars) and 16 other non-Salmonella bacteria strains were used to evaluate the specificity of the MAb by direct Slide Agglutination Test (SAT). Results showed that MAb 3E2 reacted with all Salmonella strains possessing flagellum and had no cross-reaction with non-flagellate Salmonella strains or other non-Salmonella bacteria strains. Sequentially, the ability to detect the presence of Salmonella in raw samples of the MAb 3E2-based SAT method was evaluated. The conventional culture-based detection method was performed as the standard reference method for detection of Salmonella. Altogether, 369 samples collected from laying hens were tested, and the results indicated that the MAb 3E2-based SAT method could specifically detect Salmonella. Furthermore, the SAT results were obtained more quickly, as compared with the standard method. As a whole, the MAbs against the tailored conserved region of Salmonella flagellin were prepared in this study, and MAb 3E2-based SAT is a promising candidate for the flagellated Salmonella spp. rapid detection.

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Development of a qPCR for the detection and quantification of Salmonella spp. in sheep feces and tissues.

Publications

Development of a qPCR for the detection and quantification of <i>Salmonella</i> spp. in sheep feces and tissues.

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