Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic

Qingwei Wang; Yihua Ren; Tian Meng; Xiufen Yang; Lin Lu; Hao Yang; Hongwei Hou; Masoud Negahdary; Yi Wan; Fengge Song; Jinghong Li

doi:10.1016/j.talanta.2023.125458

Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic

Talanta. 2024 Mar 1:269:125458. doi: 10.1016/j.talanta.2023.125458. Epub 2023 Nov 22.

Authors

Qingwei Wang¹, Yihua Ren², Tian Meng², Xiufen Yang², Lin Lu², Hao Yang², Hongwei Hou³, Masoud Negahdary⁴, Yi Wan⁵, Fengge Song⁶, Jinghong Li⁷

Affiliations

¹ Institute of Biomedical Engineering, College of Life Sciences, Qingdao University, Qingdao, 266071, China.
² State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228, China.
³ China National Tobacco Quality Supervision & Test Center, Zhengzhou 450001, China, Beijing Institute of Life Science and Technology, Beijing, China.
⁴ Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, 05508-000, Brazil.
⁵ State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228, China. Electronic address: 993602@hainanu.edu.cn.
⁶ State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228, China. Electronic address: songfg@hainanu.edu.cn.
⁷ Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, 100084, China.

PMID: 38008027
DOI: 10.1016/j.talanta.2023.125458

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.

Keywords: CRISPR/Cas14a1; LAMP; Pathogenic bacteria; Rapid genomic DNA extraction; Visual detection.

MeSH terms

Biological Assay
DNA, Bacterial
Gold*
Metal Nanoparticles*
Reproducibility of Results

Substances

Gold
DNA, Bacterial