Li Yanan, Xu Ru, Quan Fenglei, Wu Yonghua, Wu Yige, Zhang Yongyuan, Liang Yan, Zhang Zhenzhong, Gao Hua, Deng Ruijie, Zhang Kaixiang, Li Jinghong
School of Pharmaceutical Sciences, Tianjian Laboratory of Advanced Biomedical Sciences, Zhengzhou University, Zhengzhou, Henan, 450001, China.
School of Pharmaceutical Sciences, Tianjian Laboratory of Advanced Biomedical Sciences, Zhengzhou University, Zhengzhou, Henan, 450001, China; Nanyang First People's Hospital, Nanyang, Henan, 473000, China.
EBioMedicine. 2025 Feb;112:105564. doi: 10.1016/j.ebiom.2025.105564. Epub 2025 Jan 24.
The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge.
Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR to be one-pot assay, CasTDR can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30 min without pre-amplification.
The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30 min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA.
Our study provides a rapid and accurate molecular diagnostic tool for point-of-care testing, epidemiological screening, and the detection of diseases associated with other RNA biomarkers with excellent single nucleotide differentiation, high sensitivity, and simplicity.
National Key Research and Development Program of China (No. 2023YFB3208302), National Natural Science Foundation of China (No. 22377110, 22034004, 82402749, 82073787, 22122409), National Key Research and Development Program of China (No. 2021YFA1200104), Henan Province Fund for Cultivating Advantageous Disciplines (No. 222301420019).
RNA病毒的广泛传播和进化，如严重急性呼吸综合征冠状病毒2（SARS-CoV-2），凸显了快速鉴定病毒亚型的重要性，尤其是在非实验室环境中。以单碱基分辨率对病毒核酸进行快速且低成本的居家检测仍然是一项挑战。
将拓扑受限的DNA环设计为Cas13a反式切割的底物，以实现等温扩增后的加速反应。通过在CRISPR RNA（crRNA）中设计合成错配和发夹结构，利用CRISPR/Cas13a区分病毒基因组中单核苷酸变异（SNV）的能力，从而能够可靠地区分不同的SARS-CoV-2变体。通过将CasTDR优化为一锅法检测，CasTDR能够在无需预扩增的情况下，在不到30分钟内检测出临床样本中仅含几份拷贝的奥密克戎及其亚变体。
该检测系统具有高灵敏度（0.1 aM）、单碱基特异性，以及快速的“样本到结果”流程的优势，整个过程仅需30分钟。在检测SARS-CoV-2临床样本及其变异株时，CasTDR的准确率达到了100%。此外，便携式信号读取设备的设计便于用户友好地解读结果。针对不同RNA病毒的检测需求，只需设计相应的crRNA即可对系统进行调整。
我们的研究为即时检测、流行病学筛查以及与其他RNA生物标志物相关疾病的检测提供了一种快速、准确的分子诊断工具，具有出色的单核苷酸区分能力、高灵敏度和简便性。
国家重点研发计划（编号：2023YFB3208302）、国家自然科学基金（编号：22377110、22034004、82402749、82073787、22122409）、国家重点研发计划（编号：2021YFA1200104）、河南省优势学科培育基金（编号：222301420019）
