Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Viruses, Clinical Samples, and Antibodies
2.2. The Expression and Purification of Cas12a Protein
2.3. Viral DNA or cDNA Preparation of Various Viruses and Clinical Samples
2.4. Design and Synthesization of crRNAs and ssDNA
2.5. Assay of Endonuclease Activity of the Purified Cas12a Proteins and Optimization of crRNAs and Cas12a Concentration
2.6. The Optimization of Recombinant Polymerase Amplification (RPA)
2.7. The Optimization of Sensitivity and Time in CRISPR/Cas12a-PCV3 Detection
2.8. The Analysis of Sensitivity and Specificity in Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection
2.9. Establishment and Sensitivity of One-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method
2.10. The Detection of Clinical Samples
2.11. Statistical Analysis
3. Results
3.1. Expression and Purification of Cas12a Protein and Assay of Endonuclease Activity
3.2. The Optimization of Cas12a and crRNA Concentration in CRISPR/Cas12a-PCV3 Detection Method
3.3. The Optimization of RPA Primers and Sensitivity of CRISPR/Cas12a-PCV3 Detection
3.4. Establishment of Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method
3.5. Sensitivity and Specificity of Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method
3.6. Evaluating Consistency between Two-Pot RPA-CRISPR/Cas12a-PCV3 Detection Method and Real-Time Quantitative PCR (qPCR) Method
3.7. The Clinical Samples Detection of One-Pot RPA-CRISPR/Cas12a-PCV3 Method
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Sample Types | Sample Numbers | RPA-CRISPR/Cas12a | qPCR |
---|---|---|---|
Spleen | 6 | 6/6 | 6/6 |
Lung | 7 | 5/7 | 5/7 |
Kidney | 2 | 1/2 | 1/2 |
Submandibular lymph node | 3 | 3/3 | 3/3 |
Inguinal lymph node | 7 | 3/7 | 3/7 |
Positive rates | 72% | 72% |
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Jiang, G.; Yang, X.; Li, Z.; Mao, J.; Zeng, P.; Wang, D.; Wu, Z.; Liu, C.; Qiu, Y.; Cui, Y.; et al. Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3. Animals 2024, 14, 2527. https://doi.org/10.3390/ani14172527
Jiang G, Yang X, Li Z, Mao J, Zeng P, Wang D, Wu Z, Liu C, Qiu Y, Cui Y, et al. Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3. Animals. 2024; 14(17):2527. https://doi.org/10.3390/ani14172527
Chicago/Turabian StyleJiang, Genghong, Xiaoyu Yang, Zhaoyang Li, Jingyu Mao, Penghui Zeng, Dedong Wang, Zhi Wu, Changzhe Liu, Yonghui Qiu, Yongqiu Cui, and et al. 2024. "Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3" Animals 14, no. 17: 2527. https://doi.org/10.3390/ani14172527