Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Stains
2.2. Enrichment Optimization
2.2.1. Media Formulation
2.2.2. Media Supplementation
2.3. Growth Kinetic Model and Statistical analysis
2.4. Food Spiking
2.5. DNA Extraction
2.5.1. Pure Culture
2.5.2. Food Samples
2.6. qPCR Reaction
2.7. Evaluation
2.7.1. Evaluation of qPCR Assay (Efficiency, Dynamic Range, Inclusivity/Exclusivity)
2.7.2. Evaluation of the Full Methodology
3. Results
3.1. Evaluation of mTA10 Medium Modifications and Supplements
3.1.1. Optimization of Buffering Agent and Temperature
3.1.2. Influence of Supplements in Mixed Cultures
3.2. Melt Peak Analysis
3.3. Evaluation of qPCR Reaction
3.3.1. Inclusivity/Exclusivity
3.3.2. Efficiency and Dynamic Range of qPCR in Simplex and Multiplex
3.4. Methodology Evaluation
3.4.1. Comparison of Expected and Obtained Results
3.4.2. LoD Determination
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Bacterial Species | Source | N | fimA | actA | rfbE | IAC |
---|---|---|---|---|---|---|
Salmonella spp. | Mollusk (AMC 90, 238, 253) | 3 | + | - | - | -* |
Salmonella spp. | Unknown (AMC 260, 261) | 2 | + | - | - | -* |
Salmonella Oranienburg | Mollusk (AMC 28) | 1 | + | - | - | -* |
Salmonella Veneziana | Mollusk (AMC 200) | 1 | + | - | - | -* |
Salmonella Anatum | Proficiency test (AMC 60) | 1 | + | - | - | -* |
Salmonella Enteritidis | Proficiency test (AMC 82) | 1 | + | - | - | -* |
Salmonella Enteritidis | University of Bristol | 1 | + | - | - | -* |
Salmonella Wentworth | Proficiency test (AMC 84) | 1 | + | - | - | -* |
Salmonella Liverpool | Proficiency test (AMC 198) | 1 | + | - | - | -* |
Salmonella Typhimurium | Proficiency test (AMC 96) | 1 | + | - | - | -* |
Salmonella Typhimurium | Unknown (WDCM 00031) | 1 | + | - | - | -* |
Listeria monocytogenes | Unknown (WDCM 00021) | 1 | - | + | - | -* |
Listeria monocytogenes | Mollusk | 8 | - | + | - | -* |
Listeria monocytogenes | Chestnut | 6 | - | + | - | -* |
Listeria monocytogenes | Chicken | 3 | - | + | - | -* |
Listeria ivanovii | WDCM 00018 | 1 | - | - | - | + |
Listeria innocua | WDCM 00017, CECT 5376, 4030, 1325, 1141, 2110 | 6 | - | - | - | + |
Staphylococcus aureus | WDCM 00034, 00033 | 2 | - | - | - | + |
Staphylococcus coagulase + | Proficiency test | 1 | - | - | - | + |
Campylobacter coli | University of Minho | 1 | - | - | - | + |
Escherichia coli | WDCM 00013, 00012 | 2 | - | - | - | + |
Escherichia coli | WDCM 00014 | 1 | - | - | + | - |
Target Microorganism | Target Gene | Primers | Sequence 5′–3′ | Concentration Used | Reference |
---|---|---|---|---|---|
L. monocytogenes | actA | actA F | TTAAGACTTGCTTTGCCAGAGAC | 900 nM | [20] |
actA R | GGTGGTGGAAATTCGAATGAGC | ||||
Salmonella spp. | fimA | fimA F | CACTAAATCCGCCGATCAAACG | 100 nM | In this study |
fimA R | TTCAGGACGATGGAGAAAGGC | ||||
E. coli O157 | rfbE | rfbE F | TCAACAGTCTTGTACAAGTCCAC | 100 nM | [21] |
rfbE R | ACTGGCCTTGTTTCGATGAG | ||||
NC-IAC * | - | NC-IAC F | TTAAGACTTGCTTTGCCAGAGAC | 100 nM | [19] |
NC-IAC R | GGTGGTGGAAATTCGAATGAGC |
30 °C | 35 °C | |||||
---|---|---|---|---|---|---|
mTA10 | mTA10-MOPS | mTA10-MOPS + Glucose | mTA10 | mTA10-MOPS | mTA10-MOPS + Glucose | |
OD600 max | 0.288 ± 0.004 | 0.261 ± 0.013 | 0.328 ± 0.010 | 0.242 ± 0.003 | 0.202 ± 0.006 | 0.292 ± 0.002 |
µmax | 0.074 ± 0.004 | 0.078 ± 0.003 | 0.088 ± 0.001 | 0.089 ± 0.017 | 0.095 ± 0.003 | 0.085 ± 0.004 |
λ | 14.166 ± 0.166 | 14.002 ± 0.157 | 14.770 ± 0.085 | 12.403 ± 0.143 | 13.090 ± 0.184 | 13.370 ± 0.079 |
Type of Sample | Contamination Level (CFU/25 g of Sample) * | N | ||||||
---|---|---|---|---|---|---|---|---|
L. monocytogenes | S. Typhimurium | E. coli O157 | actA | fimA | rfbE | IAC | ||
Infant Milk LoD | 16.6 | 7 | 6 | 6 | + | + | + | - |
10.2 | 6 | 5 | 6 | + | + | + | - | |
5 | 3 | 2 | 6 | + | + | + | - | |
2 | 1 | 1 | 6 | + (2ND) | + (3ND) | + | - | |
2 | <1 | <1 | 6 | + | + | +(3ND) | - | |
Infant Milk | - | - | - | 4 | - | - | - | + |
9 | 6 | 4 | 2 | + | + | + | - | |
9.1 | 13 | 8 | 1 | + | + | + | - | |
9 | 6 | 1 | + | + | - | - | ||
6 | 4 | 1 | - | + | + | - | ||
9 | 4 | 1 | + | - | + | - | ||
91 | 1.3 × 103 | 1 | + | + | - | - | ||
9 | 8.0 × 102 | 1 | + | - | + | - | ||
1.3 × 104 | 8.0 × 103 | 1 | - | + | + | - | ||
9.1 × 103 | 1.3 × 104 | 8.0 × 103 | 1 | + | + | + | - |
Microorganism | Target | N | PA | PD | NA | ND | SE | SP | AC | PPV | NPV | k |
---|---|---|---|---|---|---|---|---|---|---|---|---|
E. coli O157 | rfbE | 44 | 32 | 0 | 12 | 0 | 100 | 100 | 100 | 100 | 100 | 1.00 |
S. Typhimurium | fimA | 44 | 26 | 0 | 18 | 0 | 100 | 100 | 100 | 100 | 100 | 1.00 |
L. monocytogenes | actA | 44 | 26 | 0 | 18 | 0 | 100 | 100 | 100 | 100 | 100 | 1.00 |
Microorganism | Target | LoD50 * | LoD95 * | ||||
---|---|---|---|---|---|---|---|
LoD | Lower Conf. Limit | Upper Conf. Limit | LoD | Lower Conf. Limit | Upper Conf. Limit | ||
E. coli O157 | rfbE | 0.129 | 0.051 | 0.327 | 0.558 | 0.220 | 1.414 |
S. Typhimurium | fimA | 0.488 | 0.257 | 0.930 | 2.111 | 1.109 | 4.018 |
L. monocytogenes | actA | 0.613 | 0.292 | 1.288 | 2.649 | 1.261 | 5.567 |
Multiplex | 0.398 | 0.253 | 0.627 | 1.721 | 1.093 | 2.710 |
Reference | Pathogens Detected | Target | Fluorescent Dye | IAC | LoD and Matrix | Enrichment Time |
---|---|---|---|---|---|---|
Skerniškytė 2016 et al. [47] | Salmonella spp., Y. ente- rocolitica, L. monocytogenes and Campyl obacter spp. Salmonella spp. Yersinia enterocolitica Listeria monocytogenes Campylobacter spp. | invA ystA hly 16S_rrna | SYTO 9 | Yes | 3.01 log CFU/mL 2.67 log CFU/mL 4.49 log CFU/mL 2.51 log CFU/mL Chicken rinses | - |
Singh 2012 et al. [48] | Listeria monocytogenes Salmonella spp. | hly invA | SYBR Green | No | 3 log CFU per mL 1 log CFU per mL Dried milk | 6 h |
Bundidamorn 2018 et al. [42] | Shiga toxin-producing Escherichia coli Salmonella spp. Listeria monocytogenes | stx1,2 invA hly | EvaGreen | No | 1 CFU/25 g raw chicken meat, raw pork, raw beef and fresh mung bean sprout | 18 h |
Kaynak 2016 et al. [49] | Listeria monocytogenes Escherichia coli O157:H7 | hly uid | EvaGreen | No | <10 cells/mL Bovine and poultry meat | 20 h |
Xiao 2014 et al. [50] | Salmonella spp. Listeria monocytogenes Staphylococcus aureus | hly, invA, Sa442 | EvaGreen | No | 5 CFU/25 g Milk, chicken, beef, pork, egg | 24 h |
This study | Listeria monocytogenes Escherichia coli O157 Salmonella spp. | actA rfbE fimA | SYBR Green | Yes | 2 CFU/25 g <1 CFU/25 g 2 CFU/25 g Infant formula | 24 h |
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Azinheiro, S.; Carvalho, J.; Prado, M.; Garrido-Maestu, A. Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula. Microorganisms 2020, 8, 1359. https://doi.org/10.3390/microorganisms8091359
Azinheiro S, Carvalho J, Prado M, Garrido-Maestu A. Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula. Microorganisms. 2020; 8(9):1359. https://doi.org/10.3390/microorganisms8091359
Chicago/Turabian StyleAzinheiro, Sarah, Joana Carvalho, Marta Prado, and Alejandro Garrido-Maestu. 2020. "Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula" Microorganisms 8, no. 9: 1359. https://doi.org/10.3390/microorganisms8091359