Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Viral Strains
2.2. Clinical Samples
2.3. Design of Primers and Probes
2.4. Extraction of Nucleic Acids
2.5. Construction of the Standard Plasmids
2.6. Optimization of the Reaction Conditions
2.7. Specificity Analysis
2.8. Sensitivity Analysis
2.9. Repeatability Analysis
2.10. Detection of the Clinical Samples
3. Results
3.1. Construction of the Standard Plasmids
3.2. Determination of the Optimal Reaction Conditions
3.3. Generation of the Standard Curves
3.4. Sensitivity Analysis
3.5. Specificity Analysis
3.6. Repeatability Analysis
3.7. Application for Detection of Clinical Samples
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer | Sequence (5′→3′) | Product Size (bp) |
---|---|---|
C-PRRSV-F | AGTTGGGAAGATTTGGCTGTTA | 234 |
C-PRRSV-R | ACCTGCTGAAACTTACGCCGCG | |
C-PRRSV-P | CY5-TCACCGCAATGCATCTTCAGGC-BHQ2 | |
HP-PRRSV-F | GTCGCGACGTGTCCCCAAGCT | 172 |
HP-PRRSV-R | GCCCATGTTCTGCGATGGT | |
HP-PRRSV-P | FAM-CACCAGTTCCTGCACCGCGTAGAACT-BHQ1 | |
NL-PRRSV-F | AACGTATTGGACACCTCTTTTG | 217 |
NL-PRRSV-R | TGGACCTAATCTTCCTGCGTGGG | |
NL-PRRSV-P | VIC-CGGTATTCCAGTCTCGAAAAGC-BHQ1 |
Multiplex cdRT-PCR | Multiplex qRT-PCR | |||
---|---|---|---|---|
Volume (μL) | Final Concentration (nM) | Volume (μL) | Final Concentration (nM) | |
qScript XLT One-Step RT-qPCR ToughMix (2×) | 12.5 | 1× | / | / |
Fluorescein sodium salt (1 μM) | 2.5 | 100 | / | / |
One-Step RT-PCR Buffer (2×) | / | / | 12.5 | 1× |
Ex Taq HS (5 μM) | / | / | 0.5 | 100 |
Primer Script RT Enzyme Mix (5 μM) | / | / | 0.5 | 100 |
C-PRRSV-F (25 μM) | 0.4 | 400 | 0.3 | 300 |
C-PRRSV-R (25 μM) | 0.4 | 400 | 0.3 | 300 |
C-PRRSV-P (25 μM) | 0.3 | 300 | 0.2 | 200 |
HP-PRRSV-F (25 μM) | 1.0 | 1000 | 0.4 | 400 |
HP-PRRSV-R (25 μM) | 1.0 | 1000 | 0.4 | 400 |
HP-PRRSV-P (25 μM) | 0.5 | 500 | 0.5 | 500 |
NL-PRRSV-F (25 μM) | 0.6 | 600 | 0.5 | 500 |
NL-PRRSV-R (25 μM) | 0.6 | 600 | 0.5 | 500 |
NL-PRRSV-P (25 μM) | 0.4 | 400 | 0.5 | 500 |
Template (total nucleic acids) | 2.5 | / | 2.5 | / |
RNase-free distilled water | Up to 25 | / | Up to 25 | / |
Plasmid | Final Concentration (copies/µL) | Intra-Assay for Repeatability | Inter-Assay for Reproducibility | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Multiplex cdPCR (copies/µL) | Multiplex qRT-PCR(Ct) | Multiplex cdPCR (copies/µL) | Multiplex qRT-PCR (Ct) | ||||||||||
SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | ||||||
p-C-PRRSV | 3.2 × 103 | 2334.33 | 37.45 | 1.68 | 24.171 | 0.05 | 0.19 | 2332.55 | 14.42 | 0.62 | 23.81 | 0.38 | 1.60 |
3.2 × 102 | 214.7 | 2.65 | 1.31 | 27.71 | 0.35 | 1.26 | 217.30 | 3.63 | 1.67 | 27.67 | 0.09 | 0.32 | |
3.2 × 101 | 19.10 | 0.25 | 1.70 | 30.89 | 0.18 | 0.58 | 18.90 | 0.38 | 2.01 | 30.67 | 0.52 | 1.70 | |
p-HP-PRRSV | 3.2 × 103 | 2324 | 39.60 | 1.70 | 25.76 | 0.10 | 0.39 | 2310 | 57.20 | 2.48 | 25.64 | 0.10 | 0.39 |
3.2 × 102 | 196.5 | 3.90 | 1.98 | 29.34 | 0.21 | 0.72 | 201.4 | 5.30 | 2.63 | 29.54 | 0.19 | 0.64 | |
3.2 × 101 | 19.46 | 0.45 | 2.31 | 32.87 | 0.28 | 0.85 | 19.13 | 0.13 | 0.68 | 32.67 | 0.22 | 0.67 | |
p-NL-PRRSV | 3.2 × 103 | 2328.3 | 37.07 | 1.59 | 24.28 | 0.26 | 1.07 | 2308.1 | 46.42 | 2.01 | 24.62 | 0.39 | 1.58 |
3.2 × 102 | 250.4 | 6.70 | 2.68 | 27.57 | 0.16 | 0.58 | 252.4 | 3.30 | 1.31 | 27.14 | 0.14 | 0.52 | |
3.2 × 101 | 23.2 | 0.55 | 2.37 | 30.92 | 0.12 | 0.39 | 22.86 | 0.57 | 2.49 | 30.74 | 0.24 | 0.78 |
Pathogen | Number | Multiplex qRT-PCR | Multiplex cdRT-PCR | Coincidence Rate (%) | Kappa | ||
---|---|---|---|---|---|---|---|
Positive | Positive Rate (%) | Positive | Positive Ratio (%) | ||||
C-PRRSV | 320 | 6 | 1.88 | 7 | 2.19 | 99.69 | 0.92 |
HP-PRRSV | 320 | 69 | 21.56 | 81 | 25.31 | 96.25 | 0.90 |
NL-PRRSV | 320 | 31 | 9.69 | 37 | 11.56 | 98.13 | 0.90 |
C-PRRSV plus HP-PRRSV | 320 | 2 | 0.63 | 2 | 0.63 | 100 | / |
C-PRRSV plus NL-PRRSV | 320 | 1 | 0.31 | 1 | 0.31 | 100 | / |
HP-PRRSV plus NL-PRRSV | 320 | 15 | 4.69 | 20 | 6.25 | 98.44 | / |
C-PRRSV plus HP-PRRSV plus NL-PRRSV | 320 | 1 | 0.31 | 1 | 0.31 | 100 | / |
Multiplex qRT-PCR | Multiplex cdRT-PCR | Coincidence Rate | Kappa Value | ||
---|---|---|---|---|---|
Positive | Negative | Total | |||
Positive | 106 | 0 | 106 | 0.941 | 0.873 |
Negative | 19 | 195 | 214 | ||
Total | 125 | 195 | 320 |
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Long, F.; Chen, Y.; Shi, K.; Yin, Y.; Feng, S.; Si, H. Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus. Animals 2023, 13, 594. https://doi.org/10.3390/ani13040594
Long F, Chen Y, Shi K, Yin Y, Feng S, Si H. Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus. Animals. 2023; 13(4):594. https://doi.org/10.3390/ani13040594
Chicago/Turabian StyleLong, Feng, Yating Chen, Kaichuang Shi, Yanwen Yin, Shuping Feng, and Hongbin Si. 2023. "Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus" Animals 13, no. 4: 594. https://doi.org/10.3390/ani13040594
APA StyleLong, F., Chen, Y., Shi, K., Yin, Y., Feng, S., & Si, H. (2023). Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus. Animals, 13(4), 594. https://doi.org/10.3390/ani13040594