Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Materials
2.2. Strain Culture and DNA Extraction
2.3. LAMP Primer Design
2.4. LAMP System Optimization
2.5. Feasibility Detection
2.6. Optimization of Dye Concentration
2.7. Selection of Gold Nanoparticle Size
2.8. Optimization of LAMP-ICTs Assay
2.9. Specificity and Sensitivity Detection in the Bacterial Solution
2.10. Detection of E. coli O157: H7 in Milk Samples
3. Results and Discussion
3.1. Primer Screening
3.2. Optimization of LAMP Reaction
3.3. Visual Detection Principle
3.4. Feasibility Detection
3.5. Selection of AuNPs with Different Particle Sizes
3.6. Optimization of ICTs Conditions
3.7. Analysis Performance for E. coli O157:H7
3.8. Analysis Performance in Milk Samples
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Primer Name | Sequence (5′-3′) | 5′-Labeled | Reference | |
---|---|---|---|---|---|
rfbE | E1 | F3 | CCACAAGGAAAGTAAAGATGTT | [23] | |
B3 | CCAACCAAGATCCTCAGC | ||||
FIP | CAAGGTGATTCCTTAATTCCTCTCTACACTTATTGGATGGTCTCA | digoxin | |||
BIP | AACTCATCGAAACAAGGCCAGGTGCTTTTGATATTTTTCCGAGTA | biotin | |||
E2 | F3 | TGGAATGGTTGTCACGAA | This study | ||
B3 | GCGATTTCACGTTTTCGT | ||||
FIP | TTGCCTATGTACAGCTAATCCTTGTTTTTGACAAAACACTTTATGACCG | digoxin | |||
BIP | ATTGGCATGACGTTATAGGCTACTTTTGCTTGTTCTAACTGGGCTAA | biotin | |||
stx2 | 21 | F3 | TCGGTGTCTGTTATTAACCA | [24] | |
B3 | TGGAAACCGTTGTCACA | ||||
FIP | AGACGAAGATGGTCAAAACGCGCAGTTATTTTGCTGTGGA | digoxin | |||
BIP | CCGGGTTCGTTAATACGGCACGGGCACTGATATATGTGT | biotin | |||
22 | F3 | CTGCTGTGACAGTGACAA | This study | ||
B3 | ACAACGGTTTCCATGACAA | ||||
FIP | TCATCATATCTGGCGTTAATGGAGTTTTCTGCTCTGGATGCATCTC | digoxin | |||
BIP | AACCAGTGAGTGACGACTGATTTTCGGACAGCAGTTATACCA | biotin | |||
LB | TTCCGGAACGTTCCAGCG |
Salmonella | E. coli O157: H7 | Listeria monocytosis | Staphylococcus aureus | Campylobacter jejuni | |
---|---|---|---|---|---|
1 | + | + | − | − | − |
2 | + | − | + | − | − |
3 | + | − | − | + | − |
4 | + | − | − | − | + |
5 | − | + | + | − | − |
6 | − | + | − | + | − |
7 | − | + | − | − | + |
8 | − | − | + | + | − |
9 | − | − | + | − | + |
10 | − | − | − | + | + |
Technique | Sample Type | Time | Limit of Detection | Reference |
---|---|---|---|---|
Fluorescence ELISA | Milk | ~5 h | 3.75 × 103 CFU mL−1 | [13] |
Colorimetric aptasensor | Milk | 250 min | 2.3 × 103 CFU mL−1 | [32] |
PCR+LFS | Carrot | 60 min | 102 CFU g−1 | [33] |
qPCR | Ground beef | ~4 h | 101 CFU mL−1 | [34] |
Photoelectrochemical biosensor | Milk, beef, fish | ~40 min | 5 CFU mL−1 | [35] |
Smartphone-based LAMP | Milk | 4 h | 101 CFU mL−1 | [36] |
LAMP+DMF | Spiked milk | 50 min | 103 CFU mL−1 | [37] |
LAMP-LFD | Lettuce | ~2 h | 101 CFU mL−1 | [23] |
Colorimetric LAMP | Milk | 40 min | 5.7 × 102 CFU mL−1 | This study |
LAMP-ICTs | Milk | 50 min | 5.7 × 102 CFU mL−1 | This study |
Group | Before (CFU mL−1) | Add (CFU mL−1) | After (CFU mL−1) | Recovery a (%) | RSD a (%) |
---|---|---|---|---|---|
1 | 0 | 0 | ND | - | - |
2 | 0 | 5.7 × 102 | 519 ± 36 | 91.1 | 6.94 |
3 | 0 | 5.7 × 103 | 5670 ± 475 | 99.5 | 8.38 |
4 | 0 | 5.7 × 104 | 58,979 ± 3359 | 103.5 | 5.70 |
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Cui, S.; Wei, Y.; Li, C.; Zhang, J.; Zhao, Y.; Peng, X.; Sun, F. Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products. Foods 2024, 13, 2143. https://doi.org/10.3390/foods13132143
Cui S, Wei Y, Li C, Zhang J, Zhao Y, Peng X, Sun F. Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products. Foods. 2024; 13(13):2143. https://doi.org/10.3390/foods13132143
Chicago/Turabian StyleCui, Shuangshuang, Yong Wei, Can Li, Jian Zhang, Yunfeng Zhao, Xiayu Peng, and Fengxia Sun. 2024. "Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products" Foods 13, no. 13: 2143. https://doi.org/10.3390/foods13132143
APA StyleCui, S., Wei, Y., Li, C., Zhang, J., Zhao, Y., Peng, X., & Sun, F. (2024). Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products. Foods, 13(13), 2143. https://doi.org/10.3390/foods13132143