Lambda exonuclease assisted helicase-dependent amplification CRISPR/Cas12a detection of Listeria monocytogenes

Chen Wang; Qingshuang Wang; Yushi Jin; Chenxi Li; Meiyan Xin; Xue Jiang; Jiayu Wan

doi:10.1016/j.biochi.2025.06.002

Lambda exonuclease assisted helicase-dependent amplification CRISPR/Cas12a detection of Listeria monocytogenes

Biochimie. 2025 Jun 9:235:106-112. doi: 10.1016/j.biochi.2025.06.002. Online ahead of print.

Authors

Chen Wang¹, Qingshuang Wang², Yushi Jin³, Chenxi Li⁴, Meiyan Xin³, Xue Jiang³, Jiayu Wan⁵

Affiliations

¹ School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, China; Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China.
² School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, China. Electronic address: wangqingshuang@163.com.
³ School of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, China.
⁴ Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China; College of Veterinary Medicine, Jilin Agricultural University, Changchun, 130118, China.
⁵ Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China. Electronic address: wanjiayu@hotmail.com.

PMID: 40499611
DOI: 10.1016/j.biochi.2025.06.002

Abstract

We describe the construction of a protospacer adjacent motif-free CRISPR/Cas12a fluorescent biosensor based on lambda exonuclease (λ-exo) and helicase-dependent amplification (HDA) to detect Listeria monocytogenes(L. monocytogenes). The hlyA gene of L. monocytogenes was amplified by HDA. After λ-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 10⁷ CFU/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.

Keywords: CRISPR/Cas12; Helicase; Isothermal amplification; Listeria monocytogenes detection.