Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Culture Preparation and DNA Extraction
2.2. RPA Primers Design
2.3. Multiple RPA Procedure
2.4. Preparation of AuNPs and Lateral Flow Dipsticks Immunoassay
2.5. Optimization of the RPA-LFIA Conditions
2.6. Specificity and Sensitivity of Multiple RPA-LFIA Assay
2.7. Evaluation of Multiple RPA-LFIA in Artificially Contaminated Food Samples
2.8. Field Samples Testing
2.9. Statistical Analysis
3. Results
3.1. Assay Principle
3.2. Establishment and Optimization of Multiple RPA-LFIA Assay
3.3. Sensitivity and Specificity of the Multiple RPA-LFIA Assay
3.4. Application of RPA-LFIA in Spiked Samples
3.5. Detection of Field Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | ID of Strains | Multiple RPA-LFIA Test Results | ||||
---|---|---|---|---|---|---|
toxR | nuc | fimY | rfbE | hlyA | ||
Vibrio parahaemolyticus | ATCC 17802 | + | - | - | - | - |
Vibrio parahaemolyticus | ATCC 33847 | + | - | - | - | - |
Vibrio parahaemolyticus | H4-3 * | + | - | - | - | - |
Vibrio parahaemolyticus | FJ14A * | + | - | - | - | - |
Vibrio parahaemolyticus | ATCC33844 | + | - | - | - | - |
Vibrio parahaemolyticus | ATCC43996 | + | - | - | - | - |
Staphylococcus aureus | ATCC 25923 | - | + | - | - | - |
Staphylococcus aureus | GIMCC 1.142 | - | + | - | - | - |
Staphylococcus aureus | CICC 10001 | - | + | - | - | - |
Staphylococcus aureus | CICC 21648 | - | + | - | - | - |
Staphylococcus aureus | ATCC 35556 | - | + | - | - | - |
Staphylococcus aureus | ATCC 4012 | - | + | - | - | - |
Salmonella enterica | ATCC 13076 *1 | - | - | + | - | - |
Salmonella enterica | GIMCC 1.345 *1 | - | - | + | - | - |
Salmonella enterica | CMCC 50041 *1 | - | - | + | - | - |
Salmonella enterica | ATCC19585 *2 | - | - | + | - | - |
Salmonella enterica | ATCC 13314 *1 | - | - | + | - | - |
Salmonella enterica | ATCC 15277 *1 | - | - | + | - | - |
Salmonella enterica | ATCC14028 *2 | - | - | + | - | - |
Escherichia coli O157:H7 | ATCC 35150 | - | - | - | + | - |
Escherichia coli O157:H7 | 61 * | - | - | - | + | - |
Escherichia coli O157:H7 | CICC 10907 | - | - | - | + | - |
Escherichia coli O157:H7 | CICC 25013 | - | - | - | + | - |
Listeria monocytogenes | ATCC 19115 *3 | - | - | - | - | + |
Listeria monocytogenes | CICC 21633 *4 | - | - | - | - | + |
Listeria monocytogenes | ATCC 19113 *3 | - | - | - | - | + |
Listeria monocytogenes | ATCC 19112 *5 | - | - | - | - | + |
Listeria monocytogenes | ATCC 43256 *4 | - | - | - | - | + |
Listeria monocytogenes | ATCC 7302 *3 | - | - | - | - | + |
Shigella flexneri | CICC 10865 | - | - | - | - | - |
Shigella sonnei | GIMCC 1.424 | - | - | - | - | - |
Legionella pneumophila | ATCC 33152 | - | - | - | - | - |
Legionella pneumophila | 07 * | - | - | - | - | - |
Vibrio cholera | GIMCC 1.449 | - | - | - | - | - |
Vibrio cholera | 007zs0902-2009 * | - | - | - | - | - |
Vibrio harveyi | ATCC 43516 | - | - | - | - | - |
Vibrio mediterranei | ATCC 43341 | - | - | - | - | - |
Vibrio vulnificus | ATCC 14100 | - | - | - | - | - |
Pseudomonas aeruginosa | ATCC 9027 | - | - | - | - | - |
Enterobacter sakazakii | ATCC 21550 | - | - | - | - | - |
Cronobacter Sakazakii | GIMCC 1.296 | - | - | - | - | - |
Cronobacter Sakazakii | CS-3 * | - | - | - | - | - |
Campylobacter jejuni subsp. | ATCC 33560 | - | - | - | - | - |
Klebsiella pneumoniae | ATCC 13883 | - | - | - | - | - |
Acinetobacter baumannii | ATCC 19606 | - | - | - | - | - |
Bacillus cereus | ATCC 9139 | - | - | - | - | - |
Enterobacter aerogenes | CICC 10293 | - | - | - | - | - |
Yersinia enterocolitica | ATCC 23715 | - | - | - | - | - |
Target Name | Target Name | Sequence (5′-3′) | Modifications | Amplification Size | Reference |
---|---|---|---|---|---|
V. parahaemolyticus | toxR-RPA F(forward primer) | TTTGTTTGGCGTGAGCAAGGTTTTGAGGTG | 5′-TET | 230 bp | [26] |
toxR-RPA R(reverse primer) | GCAGAGGCGTCATTGTTATCAGAAGCAGGT | 5′-Digoxin | |||
S. aureus | nuc-RPA F(forward primer) | CTTATAGGGATGGCTATCAGTAATGTTTCG | 5′-FAM | 158 bp | |
nuc-RPA R(reverse primer) | CCACTTCTATTTACGCCGTTATCTGTTTGT | 5′-Digoxin | |||
S. enterica | fimY-RPA F(forward primer) | TATCAGATAAAACCTCCGCTATAACACAGT | 5′-TAMRA | 133 bp | |
fimY-RPA R(reverse primer) | CTTTCCGATAAGCGAGGTTTGGAGGCTGAT | 5′-Digoxin | |||
E. coli O157:H7 | rfbE-RPA F(forward primer) | TATCTGCAAGGTGATTCCTTGATGGTCTCA | 5′-Biotin | 176 bp | [27] |
rfbE-RPA R(reverse primer) | AGGCCAGTTACCATCCTCAGCTATAGGGTG | 5′-Digoxin | |||
L. monocytogenes | hlyA-RPA F(forward primer) | CGATCACTCTGGAGGATACGTTGCTCAATT | 5′-Cy5 | 154 bp | |
hlyA-RPA R(reverse primer) | TTACCAGGCAAATAGATGGACGATGTGAAA | 5′-Digoxin |
Samples | No. of Samples | V. parahaemolyticus | S. aureus | S. enterica | E. coli O157:H7 | L. monocytogenes | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
RPA-LFIA | BAM | RPA-LFIA | BAM | RPA-LFIA | BAM | RPA-LFIA | BAM | RPA-LFIA | BAM | ||
Milk | 12 | - | - | - | - | - | - | - | - | - | - |
Raw pork | 10 | - | - | - | - | - | - | 1 | 1 | - | - |
Eggs | 9 | - | - | - | - | - | - | - | - | - | - |
Chicken | 9 | - | - | - | - | - | - | - | - | - | - |
Cheese | 9 | - | - | - | - | - | - | - | - | - | - |
Raw shrimp | 8 | 3 | 3 | - | - | 2 | 2 | - | - | - | - |
Fish | 7 | - | - | - | - | - | - | - | - | - | - |
Codfish | 6 | - | - | - | - | - | - | - | - | - | - |
Broccoli | 5 | - | - | - | - | - | - | - | - | - | - |
Fruit juice | 5 | - | - | - | - | - | - | - | - | - | - |
Total | 80 | 3 | 3 | - | - | 2 | 2 | 1 | 1 | - | - |
Positive Detection rate | / | 3.75% | 3.75% | 0% | 0% | 2.50% | 2.50% | 1.25% | 1.25% | 0% | 0% |
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Jin, B.; Ma, B.; Li, J.; Hong, Y.; Zhang, M. Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay. Microorganisms 2022, 10, 1352. https://doi.org/10.3390/microorganisms10071352
Jin B, Ma B, Li J, Hong Y, Zhang M. Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay. Microorganisms. 2022; 10(7):1352. https://doi.org/10.3390/microorganisms10071352
Chicago/Turabian StyleJin, Bei, Biao Ma, Jiali Li, Yi Hong, and Mingzhou Zhang. 2022. "Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay" Microorganisms 10, no. 7: 1352. https://doi.org/10.3390/microorganisms10071352