Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Design of CRISPR RNA (crRNA)
2.2. Development of the RT-LAMP-Coupled CRISPR–Cas12 Assays
2.2.1. RT-LAMP Reaction
2.2.2. CRISPR–Cas12 Assay with Lateral Flow-Based Readout
2.2.3. CRISPR–Cas12 Assay with Fluorescence-Based Readout
2.3. Evaluation with Clinical Samples
2.4. Ethical and Biosafety Statements
2.5. Statistical Analysis
3. Results
3.1. Optimization of the CRISPR–Cas12 Assay Conditions with Lateral Flow-Based Readout for HCV RNA Detection
3.2. Clinical Evaluation of RT-LAMP-Coupled CRISPR–Cas12 Assay for HCV RNA Detection
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Assays | RT-LAMP-Coupled CRISPR–Cas12 Assay | ||||||||
---|---|---|---|---|---|---|---|---|---|
(1st Round) | (2nd Round) | ||||||||
Lateral Flow-Based Readout | Fluorescence-Based Readout | Lateral Flow-Based Readout | Fluorescence-Based Readout | ||||||
Positive (%) | Negative (%) | Positive (%) | Negative (%) | Positive (%) | Negative (%) | Positive (%) | Negative (%) | ||
Real-time RT-PCR | Detectable (N = 100 a) | 93 (93) | 7 (7) | 93 (93) | 7 (7) | 96 (96) | 4 (4) | 96 (96) | 4 (4) |
Undetectable (N = 30 b) | 0 (0) | 30 (100) | 0 (0) | 30 (100) | ND | ND | ND | ND |
HCV Viral Load (Log10 IU/mL) | All Samples | Genotype 1 | Genotype 3 | Genotype 4 | Genotype 6 |
---|---|---|---|---|---|
n/N (%) | n/N (%) | n/N (%) | n/N (%) | n/N (%) | |
7.01–8.00 | 15/15 (100) | 7/7 (100) | 5/5 (100) | NA | 3/3 (100) |
6.01–7.00 | 34/35 (97) | 15/16 (94) | 12/12 (100) | NA | 7/7 (100) |
5.01–6.00 | 24/24 (100) | 10/10 (100) | 10/10 (100) | 1/1 (100) | 3/3 (100) |
4.01–5.00 | 21/23 (91) | 9/10 (90) | 10/11 (91) | NA | 2/2 (100) |
3.01–4.00 | 2/3 (67) | 2/2 (100) | 0/1 (0) | NA | NA |
Total | 96/100 (96) | 43/45 (96) | 37/39 (95) | 1/1 (100) | 15/15 (100) |
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Kham-Kjing, N.; Ngo-Giang-Huong, N.; Tragoolpua, K.; Khamduang, W.; Hongjaisee, S. Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay. Diagnostics 2022, 12, 1524. https://doi.org/10.3390/diagnostics12071524
Kham-Kjing N, Ngo-Giang-Huong N, Tragoolpua K, Khamduang W, Hongjaisee S. Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay. Diagnostics. 2022; 12(7):1524. https://doi.org/10.3390/diagnostics12071524
Chicago/Turabian StyleKham-Kjing, Nang, Nicole Ngo-Giang-Huong, Khajornsak Tragoolpua, Woottichai Khamduang, and Sayamon Hongjaisee. 2022. "Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay" Diagnostics 12, no. 7: 1524. https://doi.org/10.3390/diagnostics12071524