Related Experiment Video
Updated: Aug 14, 2025
Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
Published on: December 23, 2022
Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with
Jie Hao1, Longfei Xie1, Tianmu Yang1
1Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China.
Abstract:
Pasteurella multocida (P. multocida) is an important pathogenic bacterium that poses a serious threat to the development of the livestock economy and human health. Currently, the existing methods for P. multocida detection are time-consuming and require complex professional operations, limiting the application of field detection. In the study, we presented a single-pot naked-eye CRISPR-Cas12a platform (Cas12a-NEye) for the detection of P. multocida. The round tube cover allowed more Cas12a detection solution to be temporarily stored than the flat cap, enabling single-pot assays and avoiding aerosol contamination. The positive samples generated obvious red using naked eye using no excitation light and the negative samples generated blue. The limit of detection (LOD) was a single copy, without cross-reactivity with other closely related bacteria. Furthermore, we validated this platform using 16 P. multocida clinical lung samples and obtained consistent results with the real-time quantitative polymerase chain reaction (qPCR) method. The entire experimental process included rapid DNA extraction (<1 h) and Cas12a-NEye assay (25 min), which was accomplished within 1.5 h. Thus, this "sample-to-answer" platform has significant potential for P. multocida detection.
Insights
A new CRISPR-Cas12a platform enables rapid, naked-eye detection of Pasteurella multocida (P. multocida). This single-pot assay achieves high sensitivity and accuracy within 1.5 hours, improving field detection capabilities.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Pasteurella multocida (P. multocida) is a significant pathogen impacting livestock and human health.
- Current P. multocida detection methods are slow, complex, and unsuitable for field use.
Purpose of the Study:
- To develop a rapid, sensitive, and user-friendly platform for P. multocida detection.
- To enable naked-eye identification of P. multocida without specialized equipment.
Main Methods:
- Development of a single-pot CRISPR-Cas12a platform (Cas12a-NEye) with a modified tube cover for enhanced assay efficiency.
- Utilizing naked-eye observation for result interpretation (red for positive, blue for negative).
- Validation using P. multocida clinical lung samples and comparison with real-time quantitative polymerase chain reaction (qPCR).
Main Results:
- The Cas12a-NEye platform demonstrated a limit of detection (LOD) of a single copy.
- No cross-reactivity was observed with other related bacteria.
- The platform achieved consistent results with qPCR when tested on clinical samples.
- The entire sample-to-answer process was completed within 1.5 hours.
Conclusions:
- The Cas12a-NEye platform offers a rapid, sensitive, and visually interpretable method for P. multocida detection.
- This technology has significant potential for field applications in livestock and public health.
- The single-pot design enhances usability and minimizes contamination risks.
More Related Videos
07:59Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System
Published on: April 25, 2025
10:16Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
Published on: August 16, 2024