Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of Positive Control
2.2. RPA Reaction
2.3. CRISPR/Cas12a-LFA Detection
2.4. Sensitivity and Specificity of RPA/Cas12a-LFA Detection
2.5. Preparation of Crude Extract of Plant Samples
2.6. PCR for Field Sample
3. Results
3.1. Screening of Primers for the RPA Reaction
3.2. Establishment of RPA-CRISPR/Cas12a-LFA Detection
3.3. Sensitivity and Specificity of the RPA-CRISPR/Cas12a-LFA Detection
3.4. Citrus Scab Detection with Infected Field Samples
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Name | Sequence (5′–3′) | Length (bp) |
---|---|---|
EF ITS F1 | AACCAACTCTTGTCTTGTGAAACCTTTGCAGT | 32 |
EF ITS F2 | CCGGGGGACCGAACCAACTCTTGTCTTGTGAAA | 33 |
EF ITS F3 | ACTCCCCACCCTTTGCTGTTGCGAATCACGTTG | 33 |
EF ITS R1 | AATACCAAGCGGGGCTTGATTGGTGAAATGAC | 32 |
EF ITS R2 | GGGGCTTGATTGGTGAAATGACGCTCGAACAGG | 33 |
crRNA | UAAUUUCUACUAAGUGUAGAUAACGCACAUGCGCCCCUUG | 40 |
Leaves | Fruits | |||
---|---|---|---|---|
PCR Positive | PCR Negative | PCR Positive | PCR Negative | |
RPA-CRISPR/Cas12a-LFA positive | 22 | 0 | 22 | 0 |
RPA-CRISPR/Cas12a-LFA negative | 0 | 2 | 0 | 2 |
PPA 1 (44 of 44 = 100%) | NPA 2 (4 of 4 = 100%) |
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Shin, K.; Kwon, S.-H.; Lee, S.-C.; Moon, Y.-E. Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay. Plants 2021, 10, 2132. https://doi.org/10.3390/plants10102132
Shin K, Kwon S-H, Lee S-C, Moon Y-E. Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay. Plants. 2021; 10(10):2132. https://doi.org/10.3390/plants10102132
Chicago/Turabian StyleShin, Kihye, Soon-Hwa Kwon, Seong-Chan Lee, and Young-Eel Moon. 2021. "Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay" Plants 10, no. 10: 2132. https://doi.org/10.3390/plants10102132