Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food

Abstract

Background: Although 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.

Results: We 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.

Conclusions: A 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.

Figure 1

Standard curves for detection of Salmonella by PMA-qPCR. Live Salmonella cells treated with PMA or without PMA (A); dead Salmonella cells treated with PMA or without PMA (B). Results were the average of three independent assays with triplicates ± standard deviation.

Figure 2

Discrimination of live Salmonella cells from live/dead cell mixtures. Dead cells at concentration of 3 × 10⁶ CFU/g were mixed with different number of live cells as indicated and treated with PMA or without PMA. Results were the average of three independent assays with triplicates ± standard deviation.

Figure 3

Detection of live Salmonella cells spiked in spinach by PMA qPCR. Spinach samples were inoculated with 3 × 10¹ CFU/g, 3 × 10² CFU/g and 3 × 10³ CFU/g of live cells, respectively (A); spinach samples were inoculated 3 × 10⁷ dead cells/g and with 3 × 10¹ CFU/g, 3 × 10² CFU/g, and 3 × 10³ CFU/g of live cells, respectively, as indicated (B). Spinach samples were incubated at 35°C up to 24 h. Incubated samples were collected at different time points and treated with PMA or without PMA before DNA extraction. Results were the average of triplicates ± standard deviation.

Figure 4

Heterogenic sequences in invA gene demonstrated among Salmonella strains by BLAST. It is more intensive at the 5′- and 3-′ ends (A). Target regions (or amplicons) in invA gene used for detection of Salmonella by PCR from previous reports were indicated with dash lines. Numbers in the invA gene are nucleotide positions of the 5′- or 3-′ ends of the amplicons in PCR detection schemes (see references in Table 3), and numbers in parentheses represent amplicon length in bp in qPCR assays (B) and conventional PCR assays (C). Subjects in the figure are not in scale.

Figure 5

The strategy used for the development of PMA-qPCR assay for detection of Salmonella. Five primer pairs were designed in the conserved region near the 5′-end of invA gene (red block, from nucleotide positions 167 to 540). All five primer pairs shared the same forward primer and probe, and the reverse primers (A, B, C, D, and E) defined the amplicon length of amplicons A through E (Figure 5A); the numbers on amplicon D represent the locations of most of the SNPs found between the sequence of amplicon D in invA gene of Salmonella Typhimurium and the available invA gene sequences in GenBank. The number in parentheses indicates the amplicon length in bp (Figure 5B). Subjects in the figure are not in scale.