Shang Zhenlin, Liu Sitong, Liu Dongxu, Pei Xiaojing, Li Shujing, He Yifan, Tong Yigang, Liu Guoqi
School of Light Industry Science and Engineering, Beijing Technology and Business University, Beijing 100048, China.
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Molecules. 2024 Dec 23;29(24):6066. doi: 10.3390/molecules29246066.
Viruses, known for causing widespread biological harm and even extinction, pose significant challenges to public health. Virus detection is crucial for accurate disease diagnosis and preventing the spread of infections. Recently, the outstanding analytical performance of CRISPR/Cas biosensors has shown great potential and they have been considered as augmenting methods for reverse-transcription polymerase chain reaction (RT-PCR), which was the gold standard for nucleic acid detection. We herein utilized Cas12a with universal CRISPR RNA (crRNA) for indiscriminate virus detection by attaching the target to a longer track strand for isothermal amplification. The amplified products contain a domain that is recognized by the Cas12a/crRNA complex, triggering the cleavage of surrounding reporters to produce signals, thereby escaping the target dependence of crRNA recognition. The proposed method allows the same crRNA to detect multiple viral nucleic acids with high sensitivity, including but not limited to , human papillomaviruses (), , , and miRNA biomarkers. Taking and pseudoviruses as examples, this method was proved as a versatile and sensitive platform for molecular diagnostic applications.
病毒以造成广泛的生物危害甚至灭绝而闻名,对公共卫生构成重大挑战。病毒检测对于准确的疾病诊断和预防感染传播至关重要。最近,CRISPR/Cas生物传感器出色的分析性能显示出巨大潜力,它们被认为是逆转录聚合酶链反应(RT-PCR)的补充方法,而RT-PCR是核酸检测的金标准。我们在此利用带有通用CRISPR RNA(crRNA)的Cas12a,通过将靶标连接到更长的跟踪链上进行等温扩增,来进行非特异性病毒检测。扩增产物包含一个被Cas12a/crRNA复合物识别的结构域,触发周围报告分子的切割以产生信号,从而摆脱了crRNA识别对靶标的依赖性。所提出的方法允许相同的crRNA以高灵敏度检测多种病毒核酸,包括但不限于人乳头瘤病毒(HPV)、、、和miRNA生物标志物。以和假病毒为例,该方法被证明是一种用于分子诊断应用的通用且灵敏的平台。