A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Viruses
2.2. Synthesis of RNA via In Vitro Transcription
2.3. Heating Unextracted Diagnostic Samples to Obliterate Nucleases (HUDSON)
2.4. Designing Primers for RPA and Guide RNAs for DETECTR
2.5. DETECTR
2.6. One-Pot DETECTR (OP DETECTR)
3. Results
3.1. Design of JEV DETECTR
3.2. Specificity of JEV DETECTR
3.3. Determination of Limit of Detection (LoD) of Conventional JEV DETECTR
3.4. Determination of LoD of OP JEV DETECTR
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Genotype | Primer | Sequence |
---|---|---|
Genotype I | JEV-GI-F | GACAGCAGCTACGTGTGCAAACAAGGCTTT |
JEV-GI-R | CCGAGGTGGTGGTTCCGTGCACGAATATGC | |
Genotype III | JEV-GIII-F | TGCCACGAGGCGTGCCTTTTACCGACCTAG |
JEV-GIII-R | TGTCCTTCTCTGGGCAGCCCTGAGTGCTTC | |
Genotype V | JEV-GV-F | GGAAGGCATTTGTGGAGTGAGATCAGTCAC |
JEV-GV-R | AATGGAGCTGGTCGGTATCTTCCTGATGGT |
Genotype | gRNA | Sequence | PAM |
---|---|---|---|
Genotype I | JEV-GI-gRNA | CCGAAAAGUCCACAUCCAUU | TTTC |
Genotype III | JEV-GIII-gRNA | ACCCCAACAGCCAAGGAAGA | TTTG |
Genotype V | JEV-GV-gRNA | UCCACCACCACACUAAGAUC | TTTG |
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Kwak, N.; Park, B.J.; Song, Y.-J. A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V. Biosensors 2023, 13, 769. https://doi.org/10.3390/bios13080769
Kwak N, Park BJ, Song Y-J. A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V. Biosensors. 2023; 13(8):769. https://doi.org/10.3390/bios13080769
Chicago/Turabian StyleKwak, Namki, Bum Ju Park, and Yoon-Jae Song. 2023. "A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V" Biosensors 13, no. 8: 769. https://doi.org/10.3390/bios13080769
APA StyleKwak, N., Park, B. J., & Song, Y. -J. (2023). A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V. Biosensors, 13(8), 769. https://doi.org/10.3390/bios13080769