生物传感器
核酸
清脆的
环介导等温扩增
效应器
肉眼
DNA
检出限
质粒
计算生物学
分子信标
生物
化学
分子生物学
寡核苷酸
色谱法
生物化学
基因
作者
Omar Mukama,Jinghua Wu,Zhiyuan Li,Qiongxin Liang,Zhijian Yi,Xuewen Lu,Yujie Liu,Yumei Liu,Muzammal Hussain,Gaelle Makafe,Jiaxin Liu,Ning Xu,Lingwen Zeng
标识
DOI:10.1016/j.bios.2020.112143
摘要
CRISPR/Cas systems have displayed remarkable potential in developing novel biosensing applications for nucleic acid detection owing to the collateral cleavage activity of Cas effector proteins (Cas12, Cas13, etc.). Despite tremendous progress in recent years, the existing CRISPR/Cas based biosensing platforms have several limitations, including reliance on proper amplification methods, expensive fluorescence detection equipment, or lateral flow biosensor (LFB). Herein, we report a simple, inexpensive, and ultrasensitive DNA probe based LFB with CRISPR/Cas and loop-mediated Isothermal Amplification (namely CIA). The concept behind this approach is a non-detectable test line on the LFB when the Cas effector protein collaterally cleaves the cognate target and an ssDNA reporter sequence. The CIA based LFB can detect as low as a single copy cloned Pseudomonas aeruginosa acyltransferase gene, 1 cfu/ml plasmid containing E. coli DH5α pure cultures, as well as clinical samples without DNA extraction/purification or advanced apparatuses. No cross-reactivity with other non-target bacteria was observed. The naked eye result readout was obtained in 15 min of LAMP amplification, 30 min of Cas12 reaction, and 5 min of LFB readout. This platform is robust and of low cost for on-site testing.
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