CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform

Shen，Jienan1,2; Chen，Zhi3,4; Xie，Ruibin2; Li，Jingfeng3,4; Liu，Chunyan6; He，Yaqing5; Ma，Xiaopeng1; Yang，Hui2; Xie，Zhongjian1

2023-10-01

10.1016/j.bios.2023.115523

Biosensors and Bioelectronics   Impact Factor & Quartile

0956-5663

1873-4235

Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, HN, HN, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.

CRISPR/Cas12a Microfluidic Respiratory viruses SARS-CoV-2 variants

English

First ; Corresponding

National Natural Science Foundation of China[12274197];China Postdoctoral Science Foundation[2022M723303];National Key Research and Development Program of China[2022YFB3207200];National Natural Science Foundation of China[62074155];National Natural Science Foundation of China[62204253];

CHEMISTRY

2-s2.0-85165940819

Scopus

1.Institute of Pediatrics,Shenzhen Children's Hospital,Clinical Medical College of Southern University of Science and Technology,Shenzhen,Guangdong,518038,China
2.Institute of Biomedical and Health Engineering,Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences,Shenzhen,Guangdong,518055,China
3.Interdisciplinary Center of High Magnetic Field Physics of Shenzhen University,College of Physics and Optoelectronic Engineering,Institute of Microscale Optoelectronics,Shenzhen University,Shenzhen,518060,China
4.Shenzhen International Institute for Biomedical Research,Shenzhen,3/F, Building 1-B, Silver Star Hi-tech Industrial Park, Longhua District,518110,China
5.Institute of Pathogenic Organism,Shenzhen Center for Disease Control and Prevention,Shenzhen,518055,China
6.Department of Dermatology,Longgang District Maternity & Child Healthcare Hospital,Shenzhen,Guangdong,518172,China

Shen，Jienan,Chen，Zhi,Xie，Ruibin,et al. CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform[J]. Biosensors and Bioelectronics,2023,237.

Shen，Jienan.,Chen，Zhi.,Xie，Ruibin.,Li，Jingfeng.,Liu，Chunyan.,...&Xie，Zhongjian.(2023).CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform.Biosensors and Bioelectronics,237.

Shen，Jienan,et al."CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform".Biosensors and Bioelectronics 237(2023).