ISSN: 1859 - 2872
Purify enzyme recombinant AaCas12b for Neisseria meningitidis detection using LAMP combined CRISPR-Cas one-pot technique
Main Article Content
Keywords
Neisseria meningitidis, AaCas12b, CRISPR
Abstract
Objective: Expression and purification of AaCas12b and its application to combine LAMP and CRISPR in one-pot to detect Neisseria meningitidis. Subject and method: E. coli BL21(λDE3) cells were transformed with plasmid BPK2014-AaCas12b encoding for AaCas12b protein. After that, Niken affinity chromatography method was used for enzyme purification. Enzyme Aacas12b applied to CRISPR/Cas combined isothermal amplify reaction LAMP for Neisseria meningitidis detecting. Result: Expression and purification of AaCas12b enzyme have 130kD and has biological ctivity. LAMP combined CRISPR/AaCas12b in one-pot assay could detect Neisseria meningitidis with sensitivity of 102 copies/reaction on 60 minutes. Conclusion: Enzyme AaCas12b in-house have activity and applicability in endonuclease systems CRISPR/Cas.
Article Details
References
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2. Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV (2015) Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell 60(3): 385-397.
3. Xu Y, Li Z (2020) CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J 18: 2401-2415. doi: 10.1016/j.csbj.2020.08.031.18: 2401-2415.
4. Strecker J, Jones S, Koopal B et al (2019) Engineering of CRISPR-Cas12b for human genome editing. Nat Commun 10(1): 212.
5. Li L, Li S, and Wang J (2018) CRISPR-Cas12b-assisted nucleic acid detection platform. bioRxiv preprint.
6. Lee D, Kim EJ, Kilgore PE, Kim SA, Takahashi H, Ohnishi M, Anh DD, Dong BQ, Kim JS, Tomono J, Miyamoto S, Notomi T, Kim DW, Seki M (2015) Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid. PLoS One 10(4): 0122922.
7. Seki M, Kilgore PE, Kim EJ, Ohnishi M, Hayakawa S, Kim DW (2018) Loop-mediated isothermal amplification methods for diagnosis of bacterial meningitis. Front Pediatr 6:57. doi: 10.3389/fped.2018.00057.
8. Teng F, Cui T, Feng G et al (2018) Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4(1): 63.
9. Simpson RJ (2006) SDS-PAGE of proteins. CSH Protoc 2006(1):pdb.prot4313. doi: 10.1101/pdb.prot4313.
10. He F (2011) Bradford Protein Assay. Bio-101: e45. DOI: 10.21769/BioProtoc.45.
11. Đào Thị Huyền, Đào Thanh Quyên, Trần Thị Thanh, Ngô Tất Trung, Lê Hữu Song (2023) Biểu hiện enzyme Bst Large Fragment tái tổ hợp và ứng dụng trong khuếch đại đẳng nhiệt phát hiện Neisseria meningitidis. Journal of 108-clinical medicine and pharmacy 18(5).
12. Zhang MS, Brunner SF, Huguenin-Dezot N, Liang AD, Schmied WH, Rogerson DT, Chin JW (2017) Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing. Nat Methods 14(7): 729-736. doi: 10.1038/nmeth.4302.
13. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2): 248-254.
14. Joung J, Ladha A, Saito M, Segel M, Bruneau R, Huang MW, Kim NG, Yu X, Li J, Walker BD, Greninger AL, Jerome KR, Gootenberg JS, Abudayyeh OO, Zhang F (2020) Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv [Preprint]:2020.05.04.20091231. doi: 10.1101/2020.05.04.20091231.
15. Qiu X, Liu X, Ma X, Wang R, Chen S, Li F, Li Z (2022) One-pot isothermal LAMP-CRISPR-based assay for Klebsiella pneumoniae detection. Microbiol Spectr 10(4):e0154522. doi: 10.1128/spectrum.01545-22.
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Published
Dec 29, 2023
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How to Cite
Huyền Đào T., SơnL. H. P., Quyên Đào T., TrungN. T., & SongL. H. (2023). Purify enzyme recombinant AaCas12b for Neisseria meningitidis detection using LAMP combined CRISPR-Cas one-pot technique. Tạp Chí Y Dược lâm sàng 108, 18(8). https://doi.org/10.52389/ydls.v18i8.2094