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.

中文翻译：

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基于CRISPR / Cas12的超灵敏，即时护理侧向生物传感器，可快速检测核酸。

由于Cas效应蛋白（Cas12，Cas13等）的附带裂解活性，CRISPR / Cas系统在开发用于核酸检测的新型生物传感应用中显示出了巨大的潜力。尽管近年来取得了巨大的进步，但现有的基于CRISPR / Cas的生物传感平台仍然存在一些局限性，包括对适当的扩增方法的依赖，昂贵的荧光检测设备或侧向流生物传感器（LFB）。在本文中，我们报道了一种基于LFB的简单，廉价且超灵敏的DNA探针，具有CRISPR / Cas和环介导的等温扩增（即CIA）。该方法背后的概念是，当Cas效应子蛋白侧切切割同源靶标和ssDNA报告基因序列时，LFB上将无法检测到测试线。基于CIA的LFB可以检测出低至单拷贝的克隆的铜绿假单胞菌酰基转移酶基因，1 cfu / ml含大肠杆菌DH5α纯培养物的质粒，以及无需DNA提取/纯化或先进仪器的临床样品。没有观察到与其他非靶标细菌的交叉反应。在15分钟的LAMP扩增，30分钟的Cas12反应和5分钟的LFB读数中获得肉眼结果读数。该平台功能强大且现场测试成本低廉。和5分钟的LFB读数。该平台功能强大且现场测试成本低廉。和5分钟的LFB读数。该平台功能强大且现场测试成本低廉。