CRISPR gel: A one-pot biosensing platform for rapid and sensitive detection of HIV viral RNA.
CRISPR gel: A one-pot biosensing platform for rapid and sensitive detection of HIV viral RNA.
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CRISPR凝胶：一种用于快速灵敏检测HIV病毒RNA的一锅法生物传感平台。
DOI：
10.1016/j.aca.2023.341258
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发表时间：
2023-04
影响因子：
6.2
通讯作者：
N. Uno;Ziyue Li;L. Avery;M. Sfeir;Changchun Liu
中科院分区：
文献类型：
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作者：
N. Uno;Ziyue Li;L. Avery;M. Sfeir;Changchun Liu
CRISPR 技术最近已成为一种强大的生物传感工具，与等温扩增（例如重组酶聚合酶扩增 (RPA)）相结合，可实现灵敏且特异的核酸检测。然而，由于等温扩增的兼容性差，将等温扩增纳入一锅系统中的 CRISPR 检测仍然是一个挑战。在这里，我们通过将逆转录重组酶聚合酶扩增（RT-RPA）反应溶液与CRISPR凝胶相结合，开发了一种用于人类免疫缺陷病毒（HIV）RNA检测的简单CRISPR凝胶生物传感平台。在我们的 CRISPR 凝胶生物传感平台中，CRISPR-Cas12a 酶被嵌入到琼脂糖凝胶中，提供了一个空间分离但与 RT-RPA 反应溶液相连的反应界面。在等温孵育过程中，RT-RPA 扩增最初发生在 CRISPR 凝胶上。当 RPA 产物充分扩增并到达 CRISPR 凝胶时，整个试管中都会发生 CRISPR 反应。借助 CRISPR 凝胶生物传感平台，我们在 30 分钟内每次测试成功检测到了多达 30 个 HIV RNA 拷贝。此外，我们通过检测 HIV 临床血浆样本验证了其临床实用性，与实时 RT-PCR 方法相比取得了优越的性能。因此，我们的一锅 CRISPR 凝胶生物传感平台展示了在护理点快速、灵敏地对 HIV 和其他病原体进行分子检测的巨大潜力。
CRISPR technology has recently emerged as a powerful biosensing tool for sensitive and specific nucleic acid detection when coupled with isothermal amplification (e.g., recombinase polymerase amplification (RPA)). However, it remains a challenge to incorporate isothermal amplification into CRISPR detection in a one-pot system due to their poor compatibility. Here, we developed a simple CRISPR gel biosensing platform for human immunodeficiency virus (HIV) RNA detection by combining reverse transcription-recombinase polymerase amplification (RT-RPA) reaction solution with a CRISPR gel. In our CRISPR gel biosensing platform, CRISPR-Cas12a enzymes are embedded into the agarose gel, providing a spatially separated but connected reaction interface with the RT-RPA reaction solution. During isothermal incubation, the RT-RPA amplification occurs initially on the CRISPR gel. When RPA products are sufficiently amplified and reach the CRISPR gel, the CRISPR reaction occurs in the whole tube. With the CRISPR gel biosensing platform, we successfully detected down to 30 copies of HIV RNA per test within 30 min. Furthermore, we validated its clinical utility by detecting HIV clinical plasma samples, achieving superior performance compared with the real-time RT-PCR method. Thus, our one-pot CRISPR gel biosensing platform demonstrates great potential for rapid and sensitive molecular detection of HIV and other pathogens at the point of care.