Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Samples
2.2. Conventional PCR Assays
2.3. Quantitative PCR (qPCR) Assays
2.4. POC Device
2.5. Sensors, Antibodies, and Biofunctionalization
2.6. Analysis Protocol
- The buffer step: The buffer used was PBS + 0.05% v/v Tween 20 + 1% w/v BSA, pH = 7.4, which flowed for 15 min at a flow rate of 30 μL/min. During this step, a signal was stabilized to establish the signal baseline.
- The sample step: The sample was diluted at a ratio of 1:1 with PBS + 0.05% v/v Tween 20 + 1% w/v BSA, pH = 7.4. It flowed for 10 min at a flow rate of 30 μL/min. Binding of the analytes on the functionalized PIC surfaces occurred during this step.
- The washing step: The buffer used was PBS + 0.05% v/v Tween 20 + 1% w/v BSA, pH = 7.4, which flowed for 15 min at a flow rate of 30 μL/min. Unbound viral particles and sample residues that could affect photonic measurements were removed at this step.
- The PIC surface regeneration step: The buffer used was 50 mM Glycine + 50% v/v Ethylene Glycol, pH = 3, which flowed for 5 min at a flow rate of 30 μL/min. During this step, PIC surfaces were regenerated by releasing the captured antigens from the antibodies.
- The final washing step: The buffer used was PBS + 0.05% v/v Tween 20, pH = 7.4, which flowed for 5 min at a flow rate of 30 μL/min. Removal of the regeneration buffer was critical for the preparation of PIC surfaces for the next experiments. In this step, BSA was excluded from the washing buffer to prevent protein accumulation in the microfluidics.
2.7. Data Fitting
2.8. Data Analysis and Shift Calculation
2.9. Validation of the Device
2.9.1. Limit of Detection—LOD
2.9.2. System Performance
2.10. Statistical Analysis
3. Results
3.1. PCR Results
3.2. Data Fitting
3.3. Limit of Detection-LOD
3.4. Performance of the System
3.5. Receiver Operating Characteristic (ROC) Curves
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
References
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Primer Set | Target | Primer Sequence (5′-3′) | Amplicon Length (bp) | Literature |
---|---|---|---|---|
PPV_Set_1 | NS1 gene | Forward: TTGGTAATGTTGGTTGCTACAATGC Reverse: ACCTGAACATATGGCTTTGAATTGG | 127 | [24] |
PPV_Set_2 | NS1 gene | Forward: AGCCAAAAATGCAAACCCCAATA Reverse: CTCCACGGCTCCAAGGCTAAAG | 142 | [25] |
PPV_Set_3 | NS1 gene | Forward: ATACAATTCTATTTCATGGGCCAGC Reverse: TATGTTCTGGTCTTTCCTCGCATC | 330 | [24] |
PCV2_Set_1 | PCV-2 Capsid protein gene | Forward: TAGGTTAGGGCTGTGGCCTT Reverse: CCGCACCTTCGGATATACTG | 263 | [26] |
PCV2_Set_2 | PCV-2 Rep gene | Forward: CACATCGAGAAAGCGAAAGGAAC Reverse: TGCGGGCCAAAAAAGGTACAGTT | 505 | [27] |
PCV2_Set_3 | PCV-2 ORF1 | Forward: GCCAGTTCGTCACCCTTTC Reverse: CTCCCGCACCTTCGGATAT | 657 | [28] |
Primer Set | Pre-Denaturation at 95 °C | Cycles | Denaturation at 94 °C | Annealing for 30 s at | Extension at 72 °C | Final Extension at 72 °C |
---|---|---|---|---|---|---|
PPV_Set_1 | 2 min | 32 | 20 s | 62 °C | 30 s | 1 min |
PPV_Set_2 | 2 min | 32 | 20 s | 59 °C | 30 s | 1 min |
PPV_Set_3 | 2 min | 32 | 20 s | 62 °C | 30 s | 1 min |
PCV2_Set_1 | 2 min | 32 | 20 s | 60 °C | 30 s | 1 min |
PCV2_Set_2 | 2 min | 32 | 20 s | 62 °C | 40 s | 1 min |
PCV2_Set_3 | 2 min | 32 | 20 s | 59 °C | 40 s | 1 min |
Positive and Negative Calibrators | Low Positive Calibrators | |||||
---|---|---|---|---|---|---|
Exp. Number | Sample Type | Negative/Positive | Viral Copies/mL | Sample Matrix | Virus | Viral Copies/mL |
1st | Oral fluids | Negative for PCV2 & PPV | 0 | Oral fluids | PPV, PCV-2 | 7 × 105 9 × 105 |
2nd | Oral fluids | Positive for PCV2 & PPV | 108 | Oral fluids | PPV, PCV-2 | 9 × 105 3 × 106 |
3rd | Oral fluids | Negative for PCV2 & PPV | 0 | Oral fluids | PPV, PCV-2 | 1.1 × 106 4 × 106 |
4th | Oral fluids | Positive for PPV | 108 | Feces | PCV-2 | 6 × 106 |
5th | Oral fluids | Negative for PCV2 & PPV | 0 | Feces | PPV, PCV-2 | 9 × 105 4 × 106 |
6th | Oral fluids | Positive for PCV2 | 108 |
Status of Samples According to PCR–PPV | ||||
---|---|---|---|---|
Positives | Negatives | Total | ||
Screening results | Positives | 59 (TP) | 24 (FP) | 83 |
Negatives | 27 (FN) | 81 (TN) | 108 | |
Total | 86 | 105 | 191 |
Status of Samples According to PCR–PCV-2 | ||||
---|---|---|---|---|
Positives | Negatives | Total | ||
Screening results | Positives | 57 (TP) | 33 (FP) | 90 |
Negatives | 25 (FN) | 78 (TN) | 103 | |
Total | 82 | 111 | 193 |
Metrics | PPV % | PCV-2 % |
---|---|---|
Sensitivity | 68.6 | 69.5 |
Specificity | 77.1 | 70.3 |
Accuracy | 73.3 | 69.9 |
Precision | 71.1 | 63.3 |
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Manessis, G.; Mourouzis, C.; Griol, A.; Zurita-Herranz, D.; Peransi, S.; Sanchez, C.; Giusti, A.; Gelasakis, A.I.; Bossis, I. Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases. Animals 2021, 11, 3193. https://doi.org/10.3390/ani11113193
Manessis G, Mourouzis C, Griol A, Zurita-Herranz D, Peransi S, Sanchez C, Giusti A, Gelasakis AI, Bossis I. Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases. Animals. 2021; 11(11):3193. https://doi.org/10.3390/ani11113193
Chicago/Turabian StyleManessis, Georgios, Christos Mourouzis, Amadeu Griol, David Zurita-Herranz, Sergio Peransi, Carlos Sanchez, Alessandro Giusti, Athanasios I. Gelasakis, and Ioannis Bossis. 2021. "Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases" Animals 11, no. 11: 3193. https://doi.org/10.3390/ani11113193