Recently, a research team led by Prof. MA Yingxin from the Shenzhen Institute of Advanced Technology (SIAT) of the Chinese Academy of Sciences developed an ultrasensitive detection method for SARS-CoV-2 nucleocapsid protein. This method couples the highly sensitive clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system with rolling circle amplification (RCA) and enzyme-linked immunosorbent assay (ELISA).  

The study was published in ACS Sensors on May 8. 

Immunoassays are a class of widely used bioanalytical techniques for detecting and quantifying target molecules. Among them, ELISA is favored for its high throughput and simple operation, making it the most commonly used technique in analyzing multiplex analytes. However, conventional ELISA still cannot detect biomarkers present in low abundance. 

"In recent years, the CRISPR system has been widely utilized in molecular diagnosis. Upon binding of CRISPR RNA with target DNA, Cas12a is activated, resulting in site-specific breaks of double-stranded DNA and nonspecific cleavage of surrounding single-stranded DNA probes, generating a fluorescent signal," said Prof. MA. 

In this study, researchers combined ELISA, RCA, and CRISPR/Cas12a to develop an ultra-sensitive immunoassay for the detection of the SARS-CoV-2 N protein. In this way, the specific interaction between the SARS-CoV-2 nucleocapsid protein and antibodies resulted in the formation of a sandwich-like immune complex on the well plate. Biotin-coupled primers bound to the immune complex and converted protein signals into nucleic acid signals through RCA amplification and the CRISPR/Cas12a system. 

Researchers found that the proposed method shows a wide linear range for the N protein, ranging from 3 fg/mL to 3 × 10⁷ fg/mL, and achieves a limit of detection as low as 1 fg/mL (15 aM, which is about nine molecules in 1 μL of the sample). The detection sensitivity was found to be four orders of magnitude higher than that of commercial ELISA kits. 

In addition, the proposed method exhibits high specificity and accuracy for the precise detection of SARS-CoV-2 pseudoviruses and clinical samples. This work greatly improves detection sensitivity and has the potential to serve as a powerful tool for highly accurate diagnosis of protein-related diseases. 

 The CRISPR/Cas12a-mediated immunoassay for ultra-sensitive detection of the SARS-CoV-2 N protein converts protein signals into nucleic acid signals. (Image by SIAT)

Media Contact:
ZHANG Xiaomin
Email:xm.zhang@siat.ac.cn