Multiple crRNAs-assisted CRISPR/Cas12a assay targeting cytochrome b gene for amplification-free detection of meat adulteration

文献类型： 外文期刊

第一作者： Chen, Yanju

作者： Chen, Yanju;Yang, Tianyi;Qian, Siwenjie;Wu, Jian;Peng, Cheng;Wang, Xiaofu;Xu, Junfeng;Ji, Feng;Wang, Tingzhang;Che, Yang;Wu, Jian

作者机构：

关键词： Clustered regularly interspaced short; palindromic repeats; Multiple crRNAs; Amplification -free; Cytochrome b; Food adulteration

期刊名称：ANALYTICA CHIMICA ACTA （ 影响因子：6.911； 五年影响因子：6.467 ）

ISSN： 0003-2670

年卷期： 2022 年 1231 卷

页码：

收录情况： SCI

摘要： Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have been widely applied in nucleic acid analysis for the high specificity. Coupled with pre-amplification steps, the sensitivity of CRISPR-based detection is greatly improved. However, an extra pre-amplification step not only complicates the detec-tion procedures but may also cause aerosol contaminations in the process of transferring amplified solution into CRISPR system. In this study, we demonstrate that combination of multiple crRNAs in CRISPR/Cas12a system can enhance the detection sensitivity. Based on it, we establish a multiple crRNAs enhanced CRISPR (meCRISPR) method and apply it to meat adulteration identification. Take cytochrome b (Cyt b) gene as a target, meCRISPR method can directly detect as low as 1.13 ng/mu L extracted pork DNA and 5% (w/w) pork contamination in pork and beef meat mixtures. There is no cross-reaction with extracted chicken, beef, duck and fish DNA. meCRISPR reaction is incubated at an isothermal temperature, and the detection process can be completed in a designed portable apparatus with a heat block, a light emitting diode and filters. For the simplicity, specificity and sufficient sensitivity of meCRISPR method, it will have great prospects in species identification, food adultera-tion, and genetically modified food detection.

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