Journal
ANALYTICA CHIMICA ACTA
Volume 1182, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.aca.2021.338943
Keywords
PIK3CA (H1047R) mutation Gene; Single-nucleotide variant (SNV); Rolling circle amplification (RCA); CRISPR; Cas 12a; Fluorescence; Allelic specific PCR (AS-PCR)
Categories
Funding
- National Natural Science Foundation of China [81772290, 81271930]
- Graduate Scientific Research and Innovation Foundation of Chongqing, China [CYB21070, CYB20070, 2020CDCGJ014]
- Fundamental Research Funds for the Central Universities [2019CDYGZD007]
- Chongqing Graduate Tutor Team Construction Project
- Chongqing University
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A method combining CRISPR/Cas12a with RCA technology was developed for ultra-sensitive and specific detection of the SNV of the PIK3CA H1047R gene. This method showed lower detection limit compared to AS-PCR, providing a new idea for medical diagnosis and laying a technical foundation for the study of PI3K/Akt/mTOR signaling pathways.
ABS T R A C T PIK3CA H1047R gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA H1047R mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA H1047R gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA H1047R gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways. (c) 2021 Elsevier B.V. All rights reserved.
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