Development and Validation of RAA-CRISPR/Cas12a-Based Assay for Detecting Porcine Rotavirus
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Plasmid, Virus Strain, and Genome
2.2. Primer Probe and crRNA Design
2.3. Preparation of PoRV Standard Plasmid
2.4. The Principle of RAA-CRISPR/Cas12a for Detection of PoRV
2.5. Establishment of an RAA-CRISPR/Cas12a Assay
2.6. Optimization of RAA-CRISPR/Cas12a Reaction Conditions
2.7. Exploration of the Detection Capability of RAA-CRISPR/Cas12a
2.7.1. Feasibility Test
2.7.2. Comparison of the Sensitivity Between RAA-CRISPR/Cas12a and qPCR
2.7.3. Specificity of RAA-CRISPR/Cas12a for Detection of PoRV
2.7.4. Assay Reproducibility of RAA-CRISPR/Cas12a for Detection of PoRV
2.7.5. Detection of Clinical Specimens
3. Results and Analysis
3.1. Screening of RAA-CRISPR/Cas12a Detection Primers and crRNA
3.2. Optimization Results of the Reaction System
3.3. Results of the Specificity Test
3.4. Results of the Sensitivity Test Comparing RAA-CRISPR/Cas12a and qPCR
3.5. Results of the Assay Reproducibility Test
3.6. Clinical Sample Detection Results of RAA-CRISPR/Cas12a and qPCR
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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Name | Primer Sequences (5′→3′) |
---|---|
PoRV-NSP3-F1 | ATGGAGTCTACTCAGCAGATGG |
PoRV-NSP3-R1 | ACCAGAATCATCCATTAC |
PoRV-crRNA1 | AAUUUCUACUAAGUGUAGAUGAAGCUGCAGUUGUUGCUGC |
PoRV-crRNA2 | AAUUUCUACUAAGUGUAGAUGAGGCUGCGGUUGUUGCUGC |
FQ-reporter | 6-FAM-TTTATTT-BHQ1 |
PoRV-RAA-F1 | ATGGAGTCTACTCAGCAGATGGTAAGCTC |
PoRV-RAA-R1 | TGAATACCCATTAATTCTAATGTTGAAGTG |
PoRV-RAA-F2 | ATGGAGTCTACTCAGCAGATGGTAAGCTCTATTAT |
PoRV-RAA-R2 | ACACCAGAGTCATCCATTACATAATCAAATTTAC |
PoRV-RAA-F3 | GGAGTCTACTCAGCAGATGGTAAGCTCTATTATTA |
PoRV-RAA-R3 | CACCAGAGTCATCCATTACATAATC |
PoRV-RAA-F4 | CGAGTCTACTCAGCAGATGGTAAGCTCT |
PoRV-RAA-R4 | CACCAGAGTCATCCATTACATAATCAAATTTACTT |
Name of Component | Dosage |
---|---|
Template plasmid | 1 μL |
Upstream and downstream primers (10 pM) | 1 μL |
2X SYBR Green Pro Taq HS Premix (Takara, Kyoto, Japan) | 10 μL |
RNase-free water | To 20 μL |
Test Evaluated (RAA-CRISPR/Cas12a) | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
Gold Standard (qPCR) | Positive | 50 | 0 | 50 |
Negative | 4 | 342 | 346 | |
Total | 54 | 342 | 396 |
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Huang, S.; Du, L.; Liu, S.; Yang, Q.; Lei, C.; Wang, H.; Yang, L.; Yang, X. Development and Validation of RAA-CRISPR/Cas12a-Based Assay for Detecting Porcine Rotavirus. Animals 2024, 14, 3387. https://doi.org/10.3390/ani14233387
Huang S, Du L, Liu S, Yang Q, Lei C, Wang H, Yang L, Yang X. Development and Validation of RAA-CRISPR/Cas12a-Based Assay for Detecting Porcine Rotavirus. Animals. 2024; 14(23):3387. https://doi.org/10.3390/ani14233387
Chicago/Turabian StyleHuang, Siyu, Longhuan Du, Song Liu, Qingcheng Yang, Changwei Lei, Hongning Wang, Liu Yang, and Xin Yang. 2024. "Development and Validation of RAA-CRISPR/Cas12a-Based Assay for Detecting Porcine Rotavirus" Animals 14, no. 23: 3387. https://doi.org/10.3390/ani14233387
APA StyleHuang, S., Du, L., Liu, S., Yang, Q., Lei, C., Wang, H., Yang, L., & Yang, X. (2024). Development and Validation of RAA-CRISPR/Cas12a-Based Assay for Detecting Porcine Rotavirus. Animals, 14(23), 3387. https://doi.org/10.3390/ani14233387