Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains
2.2. Primers and Probes
2.3. RPA-LFS
2.4. Preparation of Spiked Food Samples
2.5. Clinical Samples
2.6. qPCR
3. Results
3.1. Principle of the Biosensor
3.2. Optimization of the Duplex RPA Reaction
3.3. Determination of Visualization Lines on the 3-Segment Strip
3.4. Specificity and Sensitivity
3.5. Clinical Sample Applications
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Species | Strain Type | Designation |
---|---|---|
Vibrio cholerae | Reference strain | ATCC 14100 |
Vibrio vulnificus | Reference strain | ATCC 27562 |
Vibrio parahemolyticus | Reference strain | ATCC 17802 |
Vibrio alginolyticus | Reference strain | ATCC 17749 |
Vibrio harveyi | Reference strain | ATCC 43516 |
Vibrio mediterranei | Reference strain | ATCC 43341 |
Vibrio shilonii | Reference strain | ATCC BAA-91 |
Vibrio splendidus | Reference strain | MCCC 1A04096 |
Vibrio mimicus | Reference strain | MCCC 1A02602 |
Vibrio ichthyoenteri | Reference strain | MCCC 1A00057 |
Vibrio campbellii | Reference strain | MCCC 1A02605 |
Vibrio chagasii | Reference strain | MCCC 1B00386 |
Vibrio fluvialis | Reference strain | MCCC 1A02761 |
Vibrio natriegens | Reference strain | MCCC 1D00129 |
Vibrio ponticus | Reference strain | MCCC 1H00061 |
Vibrio rotiferianus | Reference strain | MCCC 1B00068 |
Salmonella Typhimurium | Reference strain | ATCC 14028 |
Listeria monocytogenes | Reference strain | ATCC 19115 |
Staphylococcus aureus | Reference strain | ATCC 6538 |
Method | Target | Primer/Probe Name | Sequence (5′-3′) | Length (bp) | Amplicon Size (bp) |
---|---|---|---|---|---|
RPA | V. cholerae | VC-F | ATCTTCAAGCTGTTCAACGGGAATATCTAA | 30 | 218 |
VC-R | Biotin-ATCAGCGACAATCGTTCAACTTTCAATGGC | 30 | |||
VC-P1 | DIG-ATCAGGCTTTGTGCATCTTGGTCGCGGTAGA [THF] TTGATCATCATAAGTTTCG-SpC3 | 51 | |||
VC-P2 | FITC-ATCAGGCTTTGTGCATCTTGGTCGCGGTAGA [THF] TTGATCATCATAAGTTTCG-SpC3 | 51 | |||
V. vulnificus | VV-F | GAGATGGATTCTTTGTATAACATTGCGT | 28 | 214 | |
VV-R | Biotin-ACGATGACGTTGGTTGTGTTTCATTATC | 28 | |||
VV-P1 | FITC-GGTGAAGTTGGCTGGTGGTTATTTTCTGAA [THF] CATGGTTGTTGAGCTC-SpC3 | 47 | |||
VV-P2 | DIG-GGTGAAGTTGGCTGGTGGTTATTTTCTGAA [THF] CATGGTTGTTGAGCTC-SpC3 | 47 | |||
qPCR | V. cholerae | VC195F | CCGTTGAGGCGAGTTTGGTGAGA | 23 | 195 |
VC195R | GTGCGCGGGTCGAAACTTATGAT | 23 | |||
V. vulnificus | gyr-vv1 | GTCCGCAGTGGAATCCTTCA | 20 | 285 | |
gyr-vv2 | TGGTTCTTACGGTTACGGCC | 20 |
No. | Food Type | Sample Source | Detection Results for V. cholerae | Detection Results for V. vulnificus | ||
---|---|---|---|---|---|---|
RPA-LFS | qPCR | RPA-LFS | qPCR | |||
1 | Shrimp | Qingdao, China | - | - | - | - |
2 | Shrimp | Qingdao, China | - | - | - | - |
3 | Shrimp | Qingdao, China | - | - | - | - |
4 | Shrimp | Qingdao, China | - | - | - | - |
5 | Shrimp | Qingdao, China | + | + | - | - |
6 | Shrimp | Qingdao, China | - | - | - | - |
7 | Shrimp | Qingdao, China | - | - | + | + |
8 | Shrimp | Qingdao, China | + | + | + | + |
9 | Shrimp | Qingdao, China | - | - | - | - |
10 | Shrimp | Qingdao, China | - | - | - | - |
11 | Shrimp | Lianyungang, China | - | - | - | - |
12 | Shrimp | Lianyungang, China | - | - | - | - |
13 | Shrimp | Lianyungang, China | - | - | - | - |
14 | Shrimp | Lianyungang, China | - | - | - | - |
15 | Shrimp | Lianyungang, China | - | - | - | - |
16 | Shrimp | Lianyungang, China | - | - | + | + |
17 | Shrimp | Lianyungang, China | - | - | - | - |
18 | Shrimp | Lianyungang, China | + | + | - | - |
19 | Shrimp | Lianyungang, China | - | - | - | - |
20 | Shrimp | Lianyungang, China | - | - | - | - |
21 | Fish | Qingdao, China | - | - | - | - |
22 | Fish | Qingdao, China | - | - | - | - |
23 | Fish | Qingdao, China | - | - | - | - |
24 | Fish | Qingdao, China | - | - | - | - |
25 | Fish | Qingdao, China | - | - | - | - |
26 | Fish | Yancheng, China | + | + | - | - |
27 | Fish | Yancheng, China | + | + | - | - |
28 | Fish | Yancheng, China | - | - | - | - |
29 | Fish | Yancheng, China | - | - | - | - |
30 | Fish | Yancheng, China | - | - | - | - |
31 | Shellfish | Qingdao, China | - | - | - | - |
32 | Shellfish | Qingdao, China | + | + | - | - |
33 | Shellfish | Qingdao, China | - | - | - | - |
34 | Shellfish | Qingdao, China | + | + | - | - |
35 | Shellfish | Qingdao, China | - | - | - | - |
36 | Shellfish | Lianyungang, China | - | - | + | + |
37 | Shellfish | Lianyungang, China | - | - | - | - |
38 | Shellfish | Lianyungang, China | - | - | - | - |
39 | Shellfish | Lianyungang, China | - | - | - | - |
40 | Shellfish | Lianyungang, China | - | - | - | - |
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Wang, P.; Liao, L.; Ma, C.; Zhang, X.; Yu, J.; Yi, L.; Liu, X.; Shen, H.; Gao, S.; Lu, Q. Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips. Biosensors 2021, 11, 151. https://doi.org/10.3390/bios11050151
Wang P, Liao L, Ma C, Zhang X, Yu J, Yi L, Liu X, Shen H, Gao S, Lu Q. Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips. Biosensors. 2021; 11(5):151. https://doi.org/10.3390/bios11050151
Chicago/Turabian StyleWang, Pei, Lei Liao, Chao Ma, Xue Zhang, Junwei Yu, Longyu Yi, Xin Liu, Hui Shen, Song Gao, and Qunwei Lu. 2021. "Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips" Biosensors 11, no. 5: 151. https://doi.org/10.3390/bios11050151