期刊
CHEMICAL SCIENCE
卷 12, 期 12, 页码 4455-4462出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/d0sc05717g
关键词
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资金
- National Key R&D Program of China [2018YFA0902600]
- National Natural Science Foundation of China [21535001, 81730051, 21761142006]
- National Natural Science Foundation of Guangdong Province [2019 ZT08Y191]
- Department of Education of Guangdong Province [2019KQNCX130]
- Shenzhen City [KQTD20190929172743294, JCYJ20200109141231365]
- Tencent Foundation through the XPLORER PRIZE
- Shenzhen Bay Laboratory [SZBL2019062801004]
This study presents a novel SNP detection method using the CRISPR system to improve universality, sensitivity, and specificity, successfully applied in clinical blood samples. This genotyping method can rapidly detect multiple genotypes in a short period of time.
Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting the protospacer-adjacent-motif (PAM) sequence, we improved the universality of the CRISPR/Cas12a based SNP detection; by removing the complementary ssDNA and introducing an additional nucleotide mismatch, we improved the sensitivity and specificity. We preloaded the CRISPR/Cas12a reagents into the point-of-care biochip for automating the process, increasing the stability and long-term storage. This biochip enables us to rapidly and conveniently detect the genotypes within 20 min. In a practical application, the CRISPR/Cas12a biochip successfully distinguished three genotypes (homozygous wild type; the homozygous mutant type; and the heterozygous mutant type) of the CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), and CYP2C19*3 (G636A, rs4986893) genes related to multiple cancers from 17 clinical blood samples. This CRISPR/Cas12a-based SNP genotyping method, being universal, accurate, and sensitive, will have broad applications in molecular diagnostics and clinical research.
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