A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Vaccine Strains and Clinical Samples
2.2. Design of Primers and Probes
2.3. Nucleic Acid Extraction
2.4. Construction of the Standard Plasmids
2.5. Optimization of the Multiplex qRT-PCR
2.6. Construction of Standard Curves
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 Standard Plasmids
3.2. Determination of the Optimal Reaction Conditions
3.3. Generation of Standard Curves
3.4. Specificity Analysis
3.5. Sensitivity Analysis
3.6. Repeatability Analysis
3.7. Detection Results of the 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/Probe | Sequence (5′→3′) | Product/bp |
---|---|---|
PEDV(N)-U | CTGGAATGAGCAAATTCGCTG | 140 |
PEDV(N)-D | CAACCCAGAAAACACCCTCAG | |
PEDV(N)-P | JOE-AGCGAATTGAACAACCTTCCAATTGGCA-BHQ1 | |
TGEV(M)-U | GCAATTCTTTGCGTTAGTGCAT | 102 |
TGEV(M)-D | AGCGTACAAATTCCCTGAAAGC | |
TGEV(M)-P | Texas Red-CTTCCTCTCGAAGGTGTGCCAACTGG-BHQ2 | |
PDCoV(M)-U | ATCGACCACATGGCTCCAA | 72 |
PDCoV(M)-D | CAGCTCTTGCCCATGTAGCTT | |
PDCoV(M)-P | FAM-CACACCAGTCGTTAAGCATGGCAAGCT-BHQ1 | |
SADS-CoV(N)-U | TACTGGTCCTCACGCAGATG | 120 |
SADS-CoV(N)-D | ACGATTGCGAACACCAAGAC | |
SADS-CoV(N)-P | Cy5-CAACAGCGACCCAATGCACACCCT-BHQ3 |
Reagent | Volume (µL) | Final Concentration (nM) |
---|---|---|
2× One-Step RT-PCR Buffer III (TaKaRa) | 10 | / |
Ex Taq HS (5 U/μL) (TaKaRa) | 0.4 | / |
PrimeScript RT Enzyme Mix II (TaKaRa) | 0.4 | / |
PEDV(N)-U (20 pmol/μL) | 0.2 | 200 |
PEDV(N)-D (20 pmol/μL) | 0.2 | 200 |
PEDV(N)-P (20 pmol/μL) | 0.3 | 300 |
TGEV(M)-U (20 pmol/μL) | 0.3 | 300 |
TGEV(M)-D (20 pmol/μL) | 0.3 | 300 |
TGEV(M)-P (20 pmol/μL) | 0.3 | 300 |
PDCoV(M)-U (20 pmol/μL) | 0.2 | 200 |
PDCoV(M)-D (20 pmol/μL) | 0.2 | 200 |
PDCoV(M)-P (20 pmol/μL) | 0.2 | 200 |
SADS-CoV(N)-U (20 pmol/μL) | 0.3 | 300 |
SADS-CoV(N)-D (20 pmol/μL) | 0.3 | 300 |
SADS-CoV(N)-P (20 pmol/μL) | 0.3 | 300 |
Nucleic acid template | 2.0 | / |
RNase-free distilled H2O | Up to 20 | / |
Plasmid | Concentration (Copies/μL) | Intra-Assay | Inter-Assay | ||||
---|---|---|---|---|---|---|---|
SD | CV (%) | SD | CV (%) | ||||
p-PEDV | 1.21 × 109 | 11.96 | 0.13 | 1.09 | 11.88 | 0.04 | 0.34 |
1.21 × 107 | 18.36 | 0.14 | 0.76 | 18.30 | 0.18 | 0.98 | |
1.21 × 105 | 24.44 | 0.06 | 0.25 | 24.52 | 0.16 | 0.65 | |
p-TGEV | 1.21 × 109 | 12.97 | 0.05 | 0.39 | 12.87 | 0.12 | 0.93 |
1.21 × 107 | 19.26 | 0.03 | 0.16 | 19.12 | 0.17 | 0.89 | |
1.21 × 105 | 25.12 | 0.19 | 0.76 | 25.10 | 0.09 | 0.36 | |
p-PDCoV | 1.21 × 109 | 11.84 | 0.10 | 0.84 | 11.72 | 0.12 | 1.02 |
1.21 × 107 | 18.27 | 0.27 | 1.48 | 18.55 | 0.10 | 0.54 | |
1.21 × 105 | 24.41 | 0.14 | 0.57 | 24.67 | 0.15 | 0.61 | |
p-SADS-CoV | 1.21 × 109 | 11.51 | 0.02 | 0.17 | 11.79 | 0.22 | 1.87 |
1.21 × 107 | 17.81 | 0.06 | 0.34 | 17.95 | 0.18 | 1.00 | |
1.21 × 105 | 24.51 | 0.35 | 1.43 | 24.75 | 0.11 | 0.44 |
Date | Number | Number of Positive Samples | |||||
---|---|---|---|---|---|---|---|
PEDV (%) | TGEV (%) | PDCoV (%) | SADS-CoV (%) | PEDV + TGEV (%) | PEDV + PDCoV (%) | ||
October, 2020 | 200 | 26 (13.00%) | 3 (1.50%) | 30 (15.00%) | 0 | 0 | 15 (7.50%) |
March, 2021 | 112 | 32 (28.57%) | 1 (0.89%) | 71 (63.39%) | 0 | 1 (0.89%) | 12 (10.71%) |
October, 2021 | 37 | 6 (16.22%) | 0 | 0 | 0 | 0 | 0 |
November, 2021 | 217 | 3 (1.38%) | 0 | 1 (0.46%) | 0 | 0 | 0 |
January, 2022 | 314 | 90 (28.66%) | 0 | 64 (20.38%) | 0 | 0 | 4 (1.27%) |
February, 2022 | 394 | 110 (27.92%) | 0 | 74 (18.78%) | 0 | 0 | 1 (0.25%) |
March, 2022 | 244 | 16 (6.56%) | 0 | 40 (16.39%) | 0 | 0 | 0 |
April, 2022 | 378 | 95 (25.13%) | 1 (0.26%) | 26 (6.88%) | 0 | 0 | 2 (0.53%) |
May, 2022 | 30 | 4 (13.33%) | 0 | 0 | 0 | 0 | 0 |
June, 2022 | 496 | 58 (11.69%) | 0 | 2 (0.40%) | 0 | 0 | 0 |
July, 2022 | 287 | 47 (16.38%) | 0 | 33 (11.50%) | 0 | 0 | 4 (1.39%) |
August, 2022 | 76 | 14 (18.42%) | 2 (2.63%) | 11 (14.47%) | 0 | 0 | 0 |
September, 2022 | 92 | 23 (25.00%) | 2 (2.17%) | 16 (17.39%) | 1 (1.09%) | 0 | 2 (2.17%) |
October, 2022 | 359 | 67 (18.66%) | 6 (1.67%) | 58 (16.16%) | 4 (1.11%) | 1 (0.28%) | 6 (1.67%) |
Total | 3236 | 591 (18.26%) | 15 (0.46%) | 426 (13.16%) | 5 (0.15%) | 2 (0.06%) | 46 (1.42%) |
Method | Positive Samples | |||
---|---|---|---|---|
PEDV (%) | TGEV (%) | PDCoV (%) | SADS-CoV (%) | |
The developed quadruplex qRT-PCR | 591/3236 (18.26%) | 15/3236 (0.46%) | 426/3236 (13.16%) | 5/3236 (0.15%) |
The reference multiplex qRT-PCR | 572/3236 (17.67%) | 15/3236 (0.46%) | 417/3236 (12.89%) | 5/3236 (0.15%) |
Agreements | 99.41% | 100% | 99.72% | 100% |
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Zhou, H.; Shi, K.; Long, F.; Zhao, K.; Feng, S.; Yin, Y.; Xiong, C.; Qu, S.; Lu, W.; Li, Z. A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses. Vet. Sci. 2022, 9, 634. https://doi.org/10.3390/vetsci9110634
Zhou H, Shi K, Long F, Zhao K, Feng S, Yin Y, Xiong C, Qu S, Lu W, Li Z. A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses. Veterinary Sciences. 2022; 9(11):634. https://doi.org/10.3390/vetsci9110634
Chicago/Turabian StyleZhou, Hongjin, Kaichuang Shi, Feng Long, Kang Zhao, Shuping Feng, Yanwen Yin, Chenyong Xiong, Sujie Qu, Wenjun Lu, and Zongqiang Li. 2022. "A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses" Veterinary Sciences 9, no. 11: 634. https://doi.org/10.3390/vetsci9110634