CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Reagents
2.2. Bacterial Strains
2.3. Clinical Sample Collection and Processing
2.4. Positive Control Preparation
2.5. Isothermal Amplification with RPA
2.6. CRISPR RNA (crRNA) Synthesis and CRISPR/Cas12a-Based Detection of ST
2.7. RPA-CRISPR/Cas12a Coupled with a Lateral Flow Detection
2.8. Establishment of Sensitivity and Specificity of LoCIST
2.9. Real-Time PCR
2.10. Bioinformatical Tools
3. Results
3.1. Construction of the Recombinant Plasmid
3.2. Establishment of the LoCIST Platform
3.3. Detection Limit of LoCIST
3.4. Clinical Specimens
3.5. Performance of the LoCIST with Clinical Samples
3.6. Unambiguous Detection of OT by cr56kDa2
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Feature | Number (n) | Percentage (%) | Total Data Availability (n) |
---|---|---|---|
Sex | |||
Male | 26 | 60.46 | 43 |
Female | 16 | 39.53 | |
Age group | |||
1.1–5 | 16 | 37.20 | 43 |
5.1–10 | 13 | 30.236 | |
10.1–15 | 7 | 16.27 | |
15.1–20 | 2 | 4.65 | |
20.1–30 | 4 | 11.62 | |
Clinical symptoms | |||
Fever Grade | |||
High | 20 | 60.6 | 33 |
Low | 7 | 21.21 | |
Vomiting | 18 | 54.54 | |
Loose motion | 5 | 15.15 | |
Abdominal pain | 7 | 21.21 | |
Headache | 9 | 27.27 | |
Retrorbital | 1 | 3.03 | |
Cough | 2 | 6.06 | |
Altered Sensorium | 15 | 45.45 | |
Seizure | 1 | 3.03 | |
Body tightening | 19 | 57.57 | |
Frothing | 12 | 36.36 | |
Up rolling of eyeball | 16 | 48.48 | |
Abnormal behavior | 1 | 3.03 | |
Time interval between symptoms onset and DOA | |||
ELISA positive | 8.75 | 16 | 40 |
ELISA negative | 5.8 | 24 |
S. No. | Platform | Isothermal Amplification | CRISPR/Cas System | End Point Detection | Gene Target | Sensitivity | Specificity (%) | LOD | Reference |
---|---|---|---|---|---|---|---|---|---|
1 | LAMP assay | LAMP | - | Agarose gel electrophoresis/real-time | groEL | - | - | 3 gene copies | [39] |
2 | 47-RPA-nfo and 47-RPA-Exo | RPA | - | Lateral flow/fluorescence | 47 kDa | 80% | 100 | 10–60 gene copies | [37] |
3 | dCas9 based SMR | RPA | dCas9 | Resonance-based micro rings | 56 kDa | 1 copy | 100 | 0.54 aM | [45] |
4 | RPA-LF | RPA | - | Lateral flow assay | 56 kDa | 100% | 90 | 10 gene copies | [38] |
5 | Real time-RPA | RPA | - | Real-time fluorescence | 47 kDa | 80% | 100 | 100 gene copies | [33] |
6 | RPA | RPA | - | Fluorescence | 56 kDa | - | - | - | [46] |
7 | SFTSV/OT/IC RT-LAMP | LAMP | - | Real-time fluorescence | - | 91.6% | 100 | 25 | [41] |
8 | LoCIST | RPA | Cas12a | Lateral flow assay | 56 kDa | 97.6% | 100 | 1 gene copy | This study |
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Bhardwaj, P.; Nanaware, N.S.; Behera, S.P.; Kulkarni, S.; Deval, H.; Kumar, R.; Dwivedi, G.R.; Kant, R.; Singh, R. CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus. Biosensors 2023, 13, 1021. https://doi.org/10.3390/bios13121021
Bhardwaj P, Nanaware NS, Behera SP, Kulkarni S, Deval H, Kumar R, Dwivedi GR, Kant R, Singh R. CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus. Biosensors. 2023; 13(12):1021. https://doi.org/10.3390/bios13121021
Chicago/Turabian StyleBhardwaj, Pooja, Nikita Shrikant Nanaware, Sthita Pragnya Behera, Smita Kulkarni, Hirawati Deval, Rajesh Kumar, Gaurav Raj Dwivedi, Rajni Kant, and Rajeev Singh. 2023. "CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus" Biosensors 13, no. 12: 1021. https://doi.org/10.3390/bios13121021