Lambda exonuclease assisted helicase-dependent amplification CRISPR/Cas12a detection of Listeria monocytogenes
文献类型： 外文期刊
作者： Wang, Chen 1 ; Wang, Qingshuang 1 ; Jin, Yushi 1 ; Li, Chenxi 2 ; Xin, Meiyan 1 ; Jiang, Xue 1 ; Wan, Jiayu 2 ;
作者机构： 1.Changchun Univ Sci & Technol, Sch Life Sci & Technol, Changchun 130022, Peoples R China
2.Chinese Acad Agr Sci, Changchun Vet Res Inst, Changchun 130122, Peoples R China
3.Jilin Agr Univ, Coll Vet Med, Changchun 130118, Peoples R China
关键词： Helicase; Isothermal amplification; CRISPR/Cas12; Listeria monocytogenes detection
期刊名称：BIOCHIMIE （ 影响因子：3.0； 五年影响因子：3.4 ）
ISSN： 0300-9084
年卷期： 2025 年 235 卷
页码：
收录情况： SCI
摘要： We describe the construction of a protospacer adjacent motif-free CRISPR/Cas12a fluorescent biosensor based on lambda exonuclease (lambda-exo) and helicase-dependent amplification (HDA) to detect Listeria monocytogenes(L. monocytogenes). The hlyA gene of L. monocytogenes was amplified by HDA. After lambda-exo catalyzed cleavage of 5 ' phosphorylated single-stranded DNA of amplification product double-stranded DNA, the double-stranded DNA formed single-stranded DNA (ssDNA). The ssDNA as a substrate activated the trans-cleavage capability of CRISPR/Cas12a to cleave the reporter gene to produce fluorescence signals. Under optimized experimental conditions, the lower limit of L. monocytogenes detection by the fluorescent biosensor was 11.5 CFU/mL, with a linear range of detection from 101 to 107CFU/mL. The fluorescent biosensor permits simple and sensitive detection of L. monocytogenes and provides a promising analysis platform for clinical diagnosis and biomedical research without protospacer adjacent motif sequence ssDNA. (c) 2025 Elsevier B.V. and Soci & eacute;t & eacute; Fran & ccedil;aise de Biochimie et Biologie Mol & eacute;culaire (SFBBM). All rights are reserved, including those for text and data mining, AI training, and similar technologies.
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