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Development of a CRISPR-Cas13a assay for mouse hepatitis virus detection
Yanhua Yang1, Tiancheng Zhang1, Ye Pan1
1School of Biological Science and Technology, Jiangsu University, Zhenjiang 212013, China.
Abstract:
Mouse hepatitis virus (MHV) is a significant pathogen that undermines the health of laboratory animals and the reliability of research data. The development of rapid and sensitive detection methods for MHV is therefore crucial for ensuring laboratory animal quality and maintaining laboratory biosafety. Here, we established a CRISPR-Cas13a-based assay targeting the E, M, and N genes of MHV by constructing a recombinase-aided amplification (RAA)-coupled detection system. Results demonstrated that the N1 target exhibited the highest sensitivity, achieving a detection limit of 0.1 copy/μL for plasmid templates and 1 copy/μL in spiked serum samples. The system also exhibited robust specificity and a strong signal response in spiked serum samples. Furthermore, we developed a multiplex amplification-free CRISPR-Cas13a assay by combining Cas13a/crRNAs targeting different genes, which achieved a detection limit of 3.06 pM. In summary, this study presents two distinct CRISPR-Cas13a-based strategies for MHV detection, demonstrating the considerable application potential of this technology for pathogen detection in laboratory animal settings.
Insights
This study developed rapid CRISPR-Cas13a assays for detecting Mouse Hepatitis Virus (MHV) in lab animals. The sensitive methods enhance animal quality and biosafety, crucial for reliable research data.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Mouse Hepatitis Virus (MHV) poses a significant threat to laboratory animal health and research integrity.
- Rapid and sensitive detection of MHV is essential for maintaining laboratory animal quality and biosafety.
Purpose of the Study:
- To establish novel CRISPR-Cas13a-based detection assays for Mouse Hepatitis Virus (MHV).
- To evaluate the sensitivity, specificity, and applicability of these assays in laboratory animal settings.
Main Methods:
- Development of a recombinase-aided amplification (RAA)-coupled CRISPR-Cas13a assay targeting MHV E, M, and N genes.
- Creation of a multiplex, amplification-free CRISPR-Cas13a assay using multiple Cas13a/crRNA targets.
- Validation using plasmid templates and spiked serum samples.
Main Results:
- The RAA-coupled CRISPR-Cas13a assay demonstrated high sensitivity, with a detection limit of 0.1 copy/μL for plasmid DNA and 1 copy/μL in serum.
- The N1 target showed the highest sensitivity in the RAA-coupled system.
- The multiplex amplification-free assay achieved a detection limit of 3.06 pM.
- Both assays exhibited robust specificity and signal response in spiked samples.
Conclusions:
- Two distinct CRISPR-Cas13a-based strategies offer effective tools for MHV detection.
- These CRISPR-Cas13a assays hold significant potential for pathogen surveillance in laboratory animal populations.
- The developed methods contribute to ensuring laboratory animal quality and enhancing biosafety protocols.
