Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Vaccine Strains and Clinical Samples
2.2. Primers and Probe
2.3. RNA Extraction and Reverse Transcription
2.4. Preparation of Standard Plasmid
2.5. Optimization of the Reaction Conditions
2.6. Generation of the Standard Curves
2.7. Specificity Analysis
2.8. Sensitivity Analysis
2.9. Repeatability Analysis
2.10. Evaluation of the Clinical Samples
3. Results
3.1. Acquirement of the Standard Plasmid
3.2. Optimization of the Reaction Conditions
3.3. Generation of Standard Curve
3.4. Analysis on the Specificity
3.5. Analysis on the Sensitivity
3.6. Analysis on the Repeatability
3.7. Evaluation of the Field Samples
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer/Probe | Sequence (5′→3′) | Product/bp |
---|---|---|
APPV-F | GGCGTGCCCAAAGAGAAAT | 90 |
APPV-R | GGCACTCTATCAAGCAGTAAGGTCTA | |
APPV-P | FAM-TCGGGTCCACCATGCCCCTTT-BHQ1 |
cdRT-PCR | qRT-PCR | |||
---|---|---|---|---|
Volume (μL) | Final Concentration (nM) | Volume (μL) | Final Concentration (nM) | |
qScript XLT One-Step RT-qPCR ToughMix (2×) (Quanta Bio., Gaithersburg, MD, USA) | 12.5 | 1× | / | / |
Fluorescein sodium salt (1000 nM) (Apexbio Bio., Beijing, China), | 2.5 | 100 | / | / |
One Step RT-PCR Buffer (2×) (TaKaRa, Dalian, China) | / | / | 12.5 | 1× |
Ex Taq HS (5000 nM) (TaKaRa, Dalian, China) | / | / | 0.5 | 100 |
Primer Script RT Enzyme Mix (5000 nM) (TaKaRa, Dalian, China) | / | / | 0.5 | 100 |
APPV-F (25,000 nM) | 0.8 | 800 | 0.4 | 400 |
APPV-R (25,000 nM) | 0.8 | 800 | 0.4 | 400 |
APPV-P (25,000 nM) | 0.4 | 400 | 0.3 | 300 |
Total nucleic acids | 2.5 | / | 2.5 | / |
Distilled water | Up to 25 | / | Up to 25 | / |
Concentration | cdRT-PCR | qRT-PCR | ||||||
---|---|---|---|---|---|---|---|---|
Intra-Assay | Inter-Assay | Intra-Assay | Inter-Assay | |||||
CV% | CV% | CV% | CV% | |||||
103 | 1114.67 ± 22.23 | 1.99 | 1121.22 ± 9.14 | 0.82 | 26.70 ± 0.24 | 0.90 | 26.64 ± 0.09 | 0.34 |
102 | 109.07 ± 4.78 | 4.38 | 108.84 ± 0.63 | 0.58 | 30.20 ± 0.18 | 0.60 | 30.23 ± 0.24 | 0.79 |
101 | 9.68 ± 0.51 | 5.27 | 9.50 ± 0.20 | 2.11 | 34.57 ± 0.25 | 0.72 | 34.42 ± 0.15 | 0.44 |
qRT-PCR | Coincidence Rate (%) | Kappa Value | ||||
---|---|---|---|---|---|---|
Positive | Negative | Total | ||||
Positive | 14 | 1 | 15 | 98.33% | 0.955 | |
cdRT-PCR | Negative | 0 | 45 | 45 | ||
Total | 14 | 46 | 60 |
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Liu, H.; Shi, K.; Feng, S.; Yin, Y.; Long, F.; Si, H. Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus. Vet. Sci. 2023, 10, 330. https://doi.org/10.3390/vetsci10050330
Liu H, Shi K, Feng S, Yin Y, Long F, Si H. Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus. Veterinary Sciences. 2023; 10(5):330. https://doi.org/10.3390/vetsci10050330
Chicago/Turabian StyleLiu, Huixin, Kaichuang Shi, Shuping Feng, Yanwen Yin, Feng Long, and Hongbin Si. 2023. "Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus" Veterinary Sciences 10, no. 5: 330. https://doi.org/10.3390/vetsci10050330