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

Li B, Chen JQ - BMC Microbiol. (2013)

Bottom Line: 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 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells by PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Biology, Center for Food Safety and Applied Nutrition, U,S, Food and Drug Administration, Laurel, MD, 20708, USA. baoguang.li@fda.hhs.gov.

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.

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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.
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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.

Mentions: Fortunately, with the usage of new high throughput sequencing platforms, many genomic sequences, including Salmonella spp., are available to the public. It has become more feasible to find specific sequences within invA gene that are highly conserved among Salmonella spp. that can be used as specific genetic markers for Salmonella spp. to detect many more Salmonella serotypes. With BLAST analysis of the invA gene sequence of Salmonella Typhimurium, we found a highly conserved segment of sequence (374 bp) near the 5′-end of the invA gene (Figure 4A), which several invA-based PCR assays have been used to target part of or the whole segment (Figure 4B;C). We took advantage of this characteristic of the invA gene to design five primer pairs in that region (Figure 5A). To enhance PMA-mediated inhibition of DNA amplification from dead cells, primer pairs were selected for one that generated high efficacy in inhibition of DNA amplification from dead cells and provided robust efficiency in DNA amplification from live cells as well. Another parameter we took into account was the compatibility between the PMA-treatment and qPCR efficiency. One study found that efficient PMA-mediated inhibition of DNA amplification required amplicons at least 190 bp in length [23]. This can be achieved when conventional PCR is in use, but amplicons longer than 190 bp might not work well in qPCR as shown in Table 1. Subsequently, an optimal amplicon (D) size of 130 bp was determined and selected for the qPCR assay development through numerous trials where PCR parameters and PMA-treatments were varied (Table 1). With amplicon D, this qPCR assay offers high sensitivity (Figure 1), and has been validated with a large number of Salmonella strains (n = 167), covering all strains from SARA (n = 72), SARB collections (n = 72) and collection strains from the recent Salmonella outbreaks (n = 23) (Additional file 1: Table S1; Table 2). All the Salmonella strains examined were positively identified without exception. This qPCR assay delivers low background on non-Salmonella strains, such as E. coli O157:H7, STEC, Shigella, or other foodborne pathogens (Table 2). The excellent performance in sensitivity and specificity is not a surprise; rather there are underlining reasons: (a) BLAST analysis of the sequence of amplicon D demonstrated that this fragment shares a remarkably high homology with most of the currently available invA sequences of Salmonella spp. It showed 100% identity with 16 genomic sequences, 99% identity (1 SNP) with 26 sequences, 98% of identity (2 SNPs) with 9 sequences, and 97% or lower identity with other sequences. (b) The positions of the mismatches with other Salmonella strains are illustrated in Figure 5B. Of the strains that showed mismatches, at least 5 strains belong to Salmonella bongori subgroup. More importantly, most of the mismatches were not located in the sequences targeted by the primers and probe we used, therefore, the changes would not affect the inclusivity of the PCR assay strategy. In contrast, numerous mismatches were found between the previously designed primer pairs listed in Table 3 and the published invA sequences of Salmonella. (c) Furthermore, we have applied this qPCR assay for detection of Salmonella from environmental water samples, which were collected and shipped to DMB lab from irrigation ponds in vegetable growing farms in southern Georgia, USA. Briefly, the water samples were concentrated by filtration, enriched with LB broth at 37°C for 24 h, purified for DNA, and subjected to this qPCR assay for detection of Salmonella. Of 150 water samples tested, over forty have been positive for Salmonella by this qPCR assay (Li et al. 2013 ASM Abstract). More significantly, we have isolated a Salmonella strain by standard culture method (FDA BAM) from every qPCR-positive (CT value under 35) water sample; and every Salmonella isolate was subsequently confirmed by traditional identification methods, and genotyped by genotyping microarray. And thus, the successful application of this qPCR assay for detection of Salmonella from irrigation water samples is testimonial for the high sensitivity and specificity of the qPCR assay (Li et al. 2013 ASM Abstract).


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

Li B, Chen JQ - BMC Microbiol. (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219372&req=5

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.
Mentions: Fortunately, with the usage of new high throughput sequencing platforms, many genomic sequences, including Salmonella spp., are available to the public. It has become more feasible to find specific sequences within invA gene that are highly conserved among Salmonella spp. that can be used as specific genetic markers for Salmonella spp. to detect many more Salmonella serotypes. With BLAST analysis of the invA gene sequence of Salmonella Typhimurium, we found a highly conserved segment of sequence (374 bp) near the 5′-end of the invA gene (Figure 4A), which several invA-based PCR assays have been used to target part of or the whole segment (Figure 4B;C). We took advantage of this characteristic of the invA gene to design five primer pairs in that region (Figure 5A). To enhance PMA-mediated inhibition of DNA amplification from dead cells, primer pairs were selected for one that generated high efficacy in inhibition of DNA amplification from dead cells and provided robust efficiency in DNA amplification from live cells as well. Another parameter we took into account was the compatibility between the PMA-treatment and qPCR efficiency. One study found that efficient PMA-mediated inhibition of DNA amplification required amplicons at least 190 bp in length [23]. This can be achieved when conventional PCR is in use, but amplicons longer than 190 bp might not work well in qPCR as shown in Table 1. Subsequently, an optimal amplicon (D) size of 130 bp was determined and selected for the qPCR assay development through numerous trials where PCR parameters and PMA-treatments were varied (Table 1). With amplicon D, this qPCR assay offers high sensitivity (Figure 1), and has been validated with a large number of Salmonella strains (n = 167), covering all strains from SARA (n = 72), SARB collections (n = 72) and collection strains from the recent Salmonella outbreaks (n = 23) (Additional file 1: Table S1; Table 2). All the Salmonella strains examined were positively identified without exception. This qPCR assay delivers low background on non-Salmonella strains, such as E. coli O157:H7, STEC, Shigella, or other foodborne pathogens (Table 2). The excellent performance in sensitivity and specificity is not a surprise; rather there are underlining reasons: (a) BLAST analysis of the sequence of amplicon D demonstrated that this fragment shares a remarkably high homology with most of the currently available invA sequences of Salmonella spp. It showed 100% identity with 16 genomic sequences, 99% identity (1 SNP) with 26 sequences, 98% of identity (2 SNPs) with 9 sequences, and 97% or lower identity with other sequences. (b) The positions of the mismatches with other Salmonella strains are illustrated in Figure 5B. Of the strains that showed mismatches, at least 5 strains belong to Salmonella bongori subgroup. More importantly, most of the mismatches were not located in the sequences targeted by the primers and probe we used, therefore, the changes would not affect the inclusivity of the PCR assay strategy. In contrast, numerous mismatches were found between the previously designed primer pairs listed in Table 3 and the published invA sequences of Salmonella. (c) Furthermore, we have applied this qPCR assay for detection of Salmonella from environmental water samples, which were collected and shipped to DMB lab from irrigation ponds in vegetable growing farms in southern Georgia, USA. Briefly, the water samples were concentrated by filtration, enriched with LB broth at 37°C for 24 h, purified for DNA, and subjected to this qPCR assay for detection of Salmonella. Of 150 water samples tested, over forty have been positive for Salmonella by this qPCR assay (Li et al. 2013 ASM Abstract). More significantly, we have isolated a Salmonella strain by standard culture method (FDA BAM) from every qPCR-positive (CT value under 35) water sample; and every Salmonella isolate was subsequently confirmed by traditional identification methods, and genotyped by genotyping microarray. And thus, the successful application of this qPCR assay for detection of Salmonella from irrigation water samples is testimonial for the high sensitivity and specificity of the qPCR assay (Li et al. 2013 ASM Abstract).

Bottom Line: 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 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells by PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Biology, Center for Food Safety and Applied Nutrition, U,S, Food and Drug Administration, Laurel, MD, 20708, USA. baoguang.li@fda.hhs.gov.

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.

Show MeSH