Yu Luxin, Tang Yuebiao, Sun Yuanzhong, Wang Houqi, Yi Hai, Zhong Yangqing, Shao Zheng, Zhou Shiqing, He Suhui, Cao Ke, Peng Lifei, Chen Zhangquan
Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, School of Medical Technology, Guangdong Medical University, Dongguan, 523808, China.
Department of Laboratory Medicine, Shenzhen Children's Hospital, Shenzhen, 518038, China.
Talanta. 2025 May 15;287:127660. doi: 10.1016/j.talanta.2025.127660. Epub 2025 Jan 27.
We present a dimethyl sulfoxide (DMSO)-enhanced one-pot HDA-CRISPR/Cas12a biosensor for the ultrasensitive detection of the monkeypox virus (MPXV). The MPXV B6R gene was initially amplified using DMSO-enhanced helicase-dependent amplification (HDA) in the bottom of the reaction tubes. DMSO was employed to enhance the amplification efficiency of HDA. CRISPR/Cas12a reagents, pre-added to the caps of the reaction tubes, were subsequently combined with HDA products to generate fluorescence signals. This DMSO-enhanced HDA-CRISPR/Cas12a biosensor enables the detection of synthetic B6R DNA within 1 hour, with a detection limit of 9 aM and a dynamic range of 10 aM to 100 pM. Our work demonstrated that 5% DMSO can enhance the sensitivity of the HDA -CRISPR/Cas12a assay by four orders of magnitude. For clinical applications, this approach can detect as low as 0.4 copies/μL of MPXV pseudovirus. A DMSO-enhanced HDA-CRISPR/Cas12a lateral flow biosensor (LFB) was developed for MPXV point-of-care testing (POCT), achieving a LOD of 10 fM. This method exhibits high specificity in distinguishing the monkeypox virus from closely related orthopoxviruses, including variola, vaccinia, cowpox, ectromelia, and camelpox. The assay is rapid (sample-to-answer times less than 1 h), cost-effective, and compatible with both fluorescence detection and the LFB for visual readouts.
我们展示了一种用于超灵敏检测猴痘病毒（MPXV）的二甲基亚砜（DMSO）增强型一锅法HDA-CRISPR/Cas12a生物传感器。最初在反应管底部使用DMSO增强的解旋酶依赖性扩增（HDA）对MPXV B6R基因进行扩增。使用DMSO来提高HDA的扩增效率。预先添加到反应管帽中的CRISPR/Cas12a试剂随后与HDA产物结合以产生荧光信号。这种DMSO增强的HDA-CRISPR/Cas12a生物传感器能够在1小时内检测合成的B6R DNA，检测限为9 aM，动态范围为10 aM至100 pM。我们的工作表明，5%的DMSO可以将HDA -CRISPR/Cas12a检测的灵敏度提高四个数量级。对于临床应用，这种方法可以检测低至0.4拷贝/μL的MPXV假病毒。开发了一种用于MPXV即时检测（POCT）的DMSO增强型HDA-CRISPR/Cas12a侧流生物传感器（LFB），检测限为10 fM。该方法在区分猴痘病毒与密切相关的正痘病毒（包括天花、痘苗、牛痘、鼠痘和骆驼痘）方面具有高特异性。该检测方法快速（样本到结果时间少于1小时）、经济高效，并且与荧光检测和用于视觉读数的LFB均兼容。
