The DOI matches its Crossref record; 35 of the 35 references it cites were found in a registry or an index.
Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology
Open accessAbstract
We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.
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Cites
A novel coronavirus from patients with pneumonia in China, 2019
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A pneumonia outbreak associated with a new coronavirus of probable bat origin
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Self-citationCRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity
Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018) · Science · doi:10.1126/science.aar6245 · Cited by 4,507
Nucleic acid detection with CRISPR-Cas13a/C2c2
Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., Verdine, V., Donghia, N., Daringer, N. M., Freije, C. A., et al. (2017) · Science · doi:10.1126/science.aam9321 · Cited by 3,844
CRISPR–Cas12-based detection of SARS-CoV-2
Broughton, J. P., Deng, X., Yu, G., Fasching, C. L., Servellita, V., Singh, J., Miao, X., Streithorst, J. A., Granados, A., Sotomayor-Gonzalez, A., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0513-4 · Cited by 2,717
Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6
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Isothermal Amplification of Nucleic Acids
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Diagnosing COVID-19: The Disease and Tools for Detection
Udugama, B., Kadhiresan, P., Kozlowski, H. N., Malekjahani, A., Osborne, M., Li, V. Y. C., Chen, H., Mubareka, S., Gubbay, J. B., & Chan, W. C. W. (2020) · ACS Nano · doi:10.1021/acsnano.0c02624 · Cited by 1,754
Cited by
Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?
Soroka, M., Wasowicz, B., & Rymaszewska, A. (2021) · Cells · doi:10.3390/cells10081931 · Cited by 473
Feng, W., Newbigging, A. M., Tao, J., Cao, Y., Peng, H., Le, C., Wu, J., Pang, B., Li, J., Tyrrell, D. L., et al. (2021) · Chemical Science · doi:10.1039/d0sc06973f · Cited by 312
Self-citationPhotocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics
Hu, M., Qiu, Z., Bi, Z., Tian, T., Jiang, Y., & Zhou, X. (2022) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2202034119 · Cited by 293
Metadata
- Authors
- Pang, B., Xu, J., Liu, Y., Peng, H., Feng, W., Cao, Y., Wu, J., Xiao, H., Pabbaraju, K., Tipples, G., Joyce, M. A., Saffran, H. A., Tyrrell, D. L., Zhang, H., & Le, X. C.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2020-11-26
- Volume
- 92
- Issue
- 24
- Pages
- 16204-16212
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- OpenAlex
- W3109667847
- Cited by
- 265
- References
- 35
- FWCI
- 4.89
- Type
- journal article
- Language
- en
- Open access
- hybrid
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- Pang, B., Xu, J., Liu, Y., Peng, H., Feng, W., Cao, Y., Wu, J., Xiao, H., Pabbaraju, K., Tipples, G., Joyce, M. A., Saffran, H. A., Tyrrell, D. L., Zhang, H., & Le, X. C. (2020). Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.0c04047
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