Liu Meiqing, Li Haoran, Jia Yanwei, Mak Pui-In, Martins Rui P
State-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau 999078, China.
Faculty of Science and Technology-ECE, University of Macau, Macau 999078, China.
Micromachines (Basel). 2021 Feb 14;12(2):197. doi: 10.3390/mi12020197.
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.
新型严重急性呼吸综合征冠状病毒2（SARS-CoV-2）作为一种人畜共患病原体的出现，导致了2019冠状病毒病（COVID-19）大流行的爆发，并给全球公共卫生带来了严重威胁。SARS-CoV-2检测的金标准方法需要先将病毒RNA逆转录（RT）为cDNA，然后进行聚合酶链反应（PCR）以扩增cDNA，这涉及多种酶、多个反应以及复杂的检测优化过程。在此，我们开发了一种基于双链特异性核酸酶（DSN）的信号放大方法，利用两种特异性DNA探针直接从病毒RNA检测SARS-CoV-2。这些特异性DNA探针可与SARS-CoV-2核衣壳蛋白基因（N基因）中不同位置的靶RNA杂交，形成DNA/RNA杂合双链。DSN切割DNA探针以释放荧光，同时使RNA链保持完整，以便与另一个可用的探针分子结合，进行进一步切割和荧光信号放大。在这项工作中，研究了优化的DSN用量、孵育温度和孵育时间。以500 pM病毒RNA的检测灵敏度证明了原理验证SARS-CoV-2检测。这种简单、快速且直接的RNA检测方法有望为检测在PCR引物结合区域发生突变的病毒提供一种补充方法，以实现更精确的检测。
