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An aM-level sensitive cascade CRISPR-Dx system (ASCas) for rapid detection of RNA without pre-amplification.
Zhang, Yibin; Chen, Yong; Zhang, Qianling; Liu, Yizhen; Zhang, Xueji.
  • Zhang Y; Research Center for Nanosensor Molecular Diagnostic & Treatment Technology, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, PR China.
  • Chen Y; Research Center for Nanosensor Molecular Diagnostic & Treatment Technology, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, PR China; Graphene Composite Research Center, College of Chemistry and Environmental Engineering, Shenzhen University,
  • Zhang Q; Graphene Composite Research Center, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, PR China.
  • Liu Y; Research Center for Nanosensor Molecular Diagnostic & Treatment Technology, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, PR China. Electronic address: yzliu@szu.edu.cn.
  • Zhang X; Research Center for Nanosensor Molecular Diagnostic & Treatment Technology, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, PR China.
Biosens Bioelectron ; 230: 115248, 2023 Jun 15.
Article in English | MEDLINE | ID: covidwho-2288684
ABSTRACT
The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acids / Biosensing Techniques / COVID-19 Type of study: Diagnostic study Limits: Humans Language: English Journal: Biosens Bioelectron Journal subject: Biotechnology Year: 2023 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acids / Biosensing Techniques / COVID-19 Type of study: Diagnostic study Limits: Humans Language: English Journal: Biosens Bioelectron Journal subject: Biotechnology Year: 2023 Document Type: Article