A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and DNA Template Preparation
2.2. Primer Design
2.3. Multiplex Reaction Protocols for RPA
2.4. Preparation of EuNP Probes
2.5. Preparation of EuNP-Based LFIA and CG–LFIA
2.6. Multiplex EuNP-Based LFIA–RPA Assay
2.7. Optimization of the Multiplex EuNP-Based LFIA–RPA Assay
2.8. Sensitivity and Specificity of the Multiplex EuNP-Based LFIA–RPA Assay
2.9. Stability of the Multiplex EuNP-Based LFIA–RPA Assay
2.10. Analysis of Spiked Samples
2.11. Evaluation of Field Food Samples
2.12. Data Analysis
3. Results
3.1. Establishment and Optimization of Multiplex EuNP-Based LFIA–RPA Assay
3.2. Sensitivity of the Multiplex EuNP-Based LFIA–RPA
3.3. Specificity of the Multiplex EuNP-Based LFIA–RPA
3.4. Stability of the Multiplex EuNP-Based LFIA–RPA
3.5. Application of the Multiplex EuNP-Based LFIA–RPA to Spiked Samples
3.6. Results of Field Food Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
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Species | ID of Strains | Results of EuNP-Based LFIA–RPA |
---|---|---|
Listeria monocytogenes | ATCC19111 | + |
Listeria monocytogenes | ATCC19115 | + |
Listeria monocytogenes | ATCC7644 | + |
Vibrio parahaemolyticus | ATCC17802 | + |
Vibrio parahaemolyticus | ATCC33847 | + |
Escherichia coli O157:H7 | GIMCC1.201 | + |
Escherichia coli O157:H7 | ECO-071 * | + |
Escherichia coli O157:H7 | ATCC35150 | + |
Legionella rubrilucens | ATCC35304 | − |
Legionella longbeachae | ATCC33462 | − |
Salmonella Enteritidis | GIMCC1.345 | − |
Salmonella Enteritidis | ATCC13076 | − |
Vibrio cholera | GIMCC1.449 | − |
Staphylococcus aureus | ATCC25923 | − |
Staphylococcus epidermidis | SE-001 * | − |
Enterobacter aerogenes | CICC10293 | − |
Enterobacter sakazalii | ATCC21550 | − |
Pseudomonas aeruginosa | GIMCC1.843 | − |
Shigella sonnei | GIMCC1.424 | − |
Salmonella cholerae | CICC21494 | − |
Shigella flexneri | CICC10865 | − |
Mycobacterium avium | CMCC93026 | − |
Target Name | Primer Name | Sequence (5′–3′) | Modifications | Amplification Size |
---|---|---|---|---|
Listeria monocytogenes (hlyA) | RPA-FP | CGATCACTCTGGAGGATACGTTGCTCAATT | 5′-Cy5 | 154 bp |
RPA-RP | TTACCAGGCAAATAGATGGACGATGTGAAA | 5′-Digoxin | ||
Vibrio parahaemolyticus (toxR) | RPA-FP | TTTGTTTGGCGTGAGCAAGGTTTTGAGGTG | 5′-Biotin | 230 bp |
RPA-RP | GCAGAGGCGTCATTGTTATCAGAAGCAGGT | 5′-Digoxin | ||
Escherichia coli O157:H7 (rfbE) | RPA-FP | TATCTGCAAGGTGATTCCTTGATGGTCTCA | 5′-FAM | 176 bp |
RPA-RP | AGGCCAGTTACCATCCTCAGCTATAGGGTG | 5′- Digoxin |
Sample (n = 8 Each) | Listeria monocytogenes | Vibrio parahaemolyticus | Escherichia coli O157:H7 | ||||||
---|---|---|---|---|---|---|---|---|---|
Spiked Concentration (CFU/mL) | Recovery of EuNP-Based LFIA (%) | Recovery of CG–LFIA (%) | Spiked Concentration (CFU/mL) | Recovery of EuNP-Based LFIA (%) | Recovery of CG–LFIA (%) | Spiked Concentration (CFU/mL) | Recovery of EuNP-Based LFIA (%) | Recovery of CG–LFIA (%) | |
Beef | 9.0 × 105 | 105.5 | 104.2 | 7.0 × 105 | 110.3 | 105.7 | 4.0 × 105 | 113.5 | 111.1 |
9.0 × 104 | 103.3 | 103.8 | 7.0 × 104 | 110.0 | 103.2 | 4.0 × 104 | 113.2 | 110.4 | |
9.0 × 103 | 101.7 | 101.5 | 7.0 × 103 | 108.5 | 101.4 | 4.0 × 103 | 112.4 | 107.5 | |
9.0 × 102 | 99.5 | 98.9 | 7.0 × 102 | 103.7 | 100.6 | 4.0 × 102 | 107.5 | 101.8 | |
9.0 × 101 | 93.1 | 93.7 | 7.0 × 101 | 99.2 | 94.1 | 4.0 × 101 | 99.6 | 96.8 | |
9.0 × 100 | 90.9 | / | 7.0 × 100 | 92.4 | / | 4.0 × 100 | 93.6 | / | |
Milk | 9.0 × 105 | 114.2 | 110.4 | 7.0 × 105 | 113.6 | 103.2 | 4.0 × 105 | 110.5 | 106.2 |
9.0 × 104 | 114.0 | 110.1 | 7.0 × 104 | 112.8 | 103.6 | 4.0 × 104 | 109.4 | 106.0 | |
9.0 × 103 | 112.9 | 108.3 | 7.0 × 103 | 111.2 | 101.5 | 4.0 × 103 | 108.8 | 104.4 | |
9.0 × 102 | 110.4 | 104.3 | 7.0 × 102 | 112.0 | 99.5 | 4.0 × 102 | 109.1 | 102.7 | |
9.0 × 101 | 97.9 | 96.1 | 7.0 × 101 | 98.6 | 94.7 | 4.0 × 101 | 94.5 | 94.6 | |
9.0 × 100 | 91.3 | / | 7.0 × 100 | 93.7 | / | 4.0 × 100 | 93.3 | / | |
Chicken breast | 9.0 × 105 | 112.1 | 106.4 | 7.0 × 105 | 111.4 | 113.2 | 4.0 × 105 | 114.1 | 112.8 |
9.0 × 104 | 111.4 | 104.8 | 7.0 × 104 | 109.8 | 113.0 | 4.0 × 104 | 113.8 | 110.4 | |
9.0 × 103 | 110.5 | 105.1 | 7.0 × 103 | 108.4 | 112.4 | 4.0 × 103 | 113.5 | 111.1 | |
9.0 × 102 | 108.1 | 103.2 | 7.0 × 102 | 106.4 | 104.7 | 4.0 × 102 | 106.5 | 106.4 | |
9.0 × 101 | 93.8 | 100.3 | 7.0 × 101 | 96.3 | 96.4 | 4.0 × 101 | 96.8 | 99.9 | |
9.0 × 100 | 92.4 | / | 7.0 × 100 | 94.1 | / | 4.0 × 100 | 93.9 | / | |
Shrimp | 9.0 × 105 | 110.8 | 109.3 | 7.0 × 105 | 113.8 | 106.3 | 4.0 × 105 | 112.0 | 104.2 |
9.0 × 104 | 109.5 | 107.7 | 7.0 × 104 | 113.2 | 104.9 | 4.0 × 104 | 111.5 | 103.9 | |
9.0 × 103 | 106.6 | 105.4 | 7.0 × 103 | 112.6 | 103.3 | 4.0 × 103 | 111.8 | 103.3 | |
9.0 × 102 | 104.8 | 102.1 | 7.0 × 102 | 109.0 | 101.1 | 4.0 × 102 | 107.3 | 99.5 | |
9.0 × 101 | 97.2 | 93.2 | 7.0 × 101 | 98.1 | 93.1 | 4.0 × 101 | 98.1 | 98.5 | |
9.0 × 100 | 94.2 | / | 7.0 × 100 | 93.4 | / | 4.0 × 100 | 95.2 | / |
Samples | Listeria monocytogenes | Vibrio parahaemolyticus | Escherichia coli O157:H7 | ||||||
---|---|---|---|---|---|---|---|---|---|
EuNP-Based LFIA–RPA | CG–LFIA–RPA | BAM | EuNP-Based LFIA–RPA | CG–LFIA–RPA | BAM | EuNP-Based LFIA–RPA | CG–LFIA–RPA | BAM | |
BF-1 | − | − | − | − | − | − | − | − | − |
BF-2 | − | − | − | − | − | − | − | − | − |
BF-3 | − | − | − | − | − | − | − | − | − |
BF-4 | − | − | − | − | − | − | − | − | − |
MK-1 | − | − | − | − | − | − | − | − | − |
MK-2 | − | − | − | − | − | − | − | − | − |
MK-3 | − | − | − | − | − | − | − | − | − |
MK-4 | − | − | − | − | − | − | − | − | − |
CB-1 | − | − | − | − | − | − | − | − | − |
CB-2 | − | − | − | − | − | − | − | − | − |
CB-3 | − | − | − | − | − | − | − | − | − |
CB-4 | − | − | − | − | − | − | − | − | − |
SP-1 | − | − | − | − | − | − | − | − | − |
SP-2 | − | − | − | − | − | − | − | − | − |
SP-3 | − | − | − | − | − | − | − | − | − |
SP-4 | − | − | − | − | − | − | − | − | − |
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Chen, K.; Ma, B.; Li, J.; Chen, E.; Xu, Y.; Yu, X.; Sun, C.; Zhang, M. A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens. Int. J. Environ. Res. Public Health 2021, 18, 4574. https://doi.org/10.3390/ijerph18094574
Chen K, Ma B, Li J, Chen E, Xu Y, Yu X, Sun C, Zhang M. A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens. International Journal of Environmental Research and Public Health. 2021; 18(9):4574. https://doi.org/10.3390/ijerph18094574
Chicago/Turabian StyleChen, Kai, Biao Ma, Jiali Li, Erjing Chen, Ying Xu, Xiaoping Yu, Chuanxin Sun, and Mingzhou Zhang. 2021. "A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens" International Journal of Environmental Research and Public Health 18, no. 9: 4574. https://doi.org/10.3390/ijerph18094574