Development of a Multiplex Quantitative PCR for Detecting Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus Simultaneously in China
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Primers and Probes
2.2. Standard Strains and Clinical Samples
2.3. Extraction of RNA and Obtained cDNA
2.4. Recombinant Plasmid Construction
2.5. Optimization of Conditions for Multiplex qPCR
2.6. Standard Curves
2.7. Specificity
2.8. Sensitivity
2.9. Repeatability
2.10. Clinical Sample Detection
3. Results
3.1. Optimization of Conditions for Multiplex qPCR
3.2. Standard Curves
3.3. Specificity
3.4. Sensitivity
3.5. Repeatability
3.6. Detection of the Clinical Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primers and probes | Sequences (5′ end to 3′ end) | Length (bp) | Use |
---|---|---|---|
PEDV-F | CCCGTTGATGAGGTGATTG | 500 | Amplification of M |
PEDV-R | TTGGCGACTGTGACGAAAT | ||
PEDV-qF | GACGCGCTTCTCACTACTTC | 134 | qPCR for the detection of M |
PEDV-qR | TGTACGCCAGTAGCAACCTT | ||
PEDV-probe | FAM-TGCAGACCTGTCGGCCCATCA-BHQ1 | ||
TGEV-F | GTCAACCCATAGCCTCAA | 497 | Amplification of S |
TGEV-R | GCCACTAAGTAGCGTCCT | ||
TGEV-qF | ACATAGTGGGTGTACCGTCTG | 140 | qPCR for the detection of S |
TGEV-qR | GCCACTAAGTAGCGTCCTGT | ||
TGEV-probe | CY5-AGCACTGACAAATCGTGCACACCA-BHQ2 | ||
PDCoV-F | TACTCATCCTCAGTTTCGTG | 598 | Amplification of N |
PDCoV-R | ACCCGTCTTCTCAGTGTCT | ||
PDCoV-qF | CAGTTTCGTGGCAATGGAGT | 79 | qPCR for the detection of N |
PDCoV-qR | TGGTGTAACGCAGCCAGTAG | ||
PDCoV-probe | HEX-CCGCTTAACTCCGCCATCAAACCCG-BHQ1 | ||
ACTB-qF | CCCTGGAGAAGAGCTACGAG | 175 | qPCR for the detection of β-Actin |
ACTB-qR | AGGTCCTTCCTGATGTCCAC | ||
ACTB-probe | Texas Red-CGGCAACGAGCGCTTCCGGT-BHQ2 |
Component | Volume (μL) |
---|---|
2 × AceQ qPCR probe master mix | 10 |
TGEV-qF/qR (10 μM) | 0.6 (0.3 μM) |
TGEV-Probe (10 μM) | 0.3 (0.15 μM) |
PEDV-qF/qR (10 μM) | 0.6 (0.3 μM) |
PEDV-Probe (10 μM) | 0.3 (0.15 μM) |
PDCoV-qF/qR (10 μM) | 0.6 (0.3 μM) |
PDCoV-Probe (10 μM) | 0.3 (0.15 μM) |
Template DNA | 2 |
ddH2O | Up to 20 |
Plasmids | Concentration (Virus copies/μL) | Intra-group | Inter-group | ||||
---|---|---|---|---|---|---|---|
Mean Ct | S.D. | CV (%) | Mean Ct | S.D. | CV (%) | ||
PEDV | 102 | 32.355 | 0.189 | 0.6 | 32.759 | 0.286 | 0.8 |
104 | 25.208 | 0.042 | 0.2 | 25.386 | 0.242 | 0.9 | |
106 | 16.418 | 0.193 | 1.2 | 16.969 | 0.431 | 2.5 | |
TGEV | 102 | 32.063 | 0.183 | 0.6 | 32.576 | 0.381 | 1.2 |
104 | 24.719 | 0.058 | 0.2 | 25.309 | 0.429 | 1.7 | |
106 | 16.421 | 0.167 | 1.0 | 17.047 | 0.452 | 2.7 | |
PDCoV | 102 | 32.361 | 0.03 | 0.0009 | 32.526 | 0.133 | 0.004 |
104 | 25.176 | 0.005 | 0.0002 | 25.428 | 0.250 | 0.009 | |
106 | 17.279 | 0.093 | 0.005 | 17.16 | 0.085 | 0.004 |
Detection Method | Number of Positive Samples | ||
---|---|---|---|
PEDV | TGEV | PDCoV | |
Multiplex qPCR | 268 | 75 | 210 |
Reference methods | 258 | 71 | 201 |
Agreements | 96.27% | 94.67% | 95.71% |
Pathogens | Number of Positive Samples | Infection Rate (%) |
---|---|---|
PEDV | 91 | 19.70 |
TGEV | 4 | 0.87 |
PDCoV | 47 | 10.17 |
PEDV/TGEV | 15 | 3.25 |
PEDV/PDCoV | 107 | 23.16 |
TGEV/PDCoV | 1 | 0.22 |
PEDV/TGEV/PDCoV | 55 | 11.90 |
β-Actin | 462 | 100 |
In total | 462 | / |
Province | Amount | Positive samples | Negative samples | ||
---|---|---|---|---|---|
PEDV | TGEV | PDCoV | |||
Jiangsu | 145 | 78 | 32 | 87 | 52 |
Guangdong | 97 | 51 | 13 | 62 | 25 |
Hubei | 92 | 53 | 6 | 16 | 34 |
Shandong | 64 | 35 | 2 | 20 | 28 |
Hunan | 64 | 51 | 23 | 25 | 3 |
Sum | 462 | 268 | 76 | 210 | 142 |
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Chen, J.; Liu, R.; Liu, H.; Chen, J.; Li, X.; Zhang, J.; Zhou, B. Development of a Multiplex Quantitative PCR for Detecting Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus Simultaneously in China. Vet. Sci. 2023, 10, 402. https://doi.org/10.3390/vetsci10060402
Chen J, Liu R, Liu H, Chen J, Li X, Zhang J, Zhou B. Development of a Multiplex Quantitative PCR for Detecting Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus Simultaneously in China. Veterinary Sciences. 2023; 10(6):402. https://doi.org/10.3390/vetsci10060402
Chicago/Turabian StyleChen, Jianpeng, Rongchao Liu, Huaicheng Liu, Jing Chen, Xiaohan Li, Jianfeng Zhang, and Bin Zhou. 2023. "Development of a Multiplex Quantitative PCR for Detecting Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus Simultaneously in China" Veterinary Sciences 10, no. 6: 402. https://doi.org/10.3390/vetsci10060402