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Updated: Jul 19, 2026
DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning
Published on: May 10, 2024
Rapid nucleic acid detection of Mycoplasma synoviae using dual-mode RAA-CRISPR/Cas12a system
Qiao Hu1, Jiaying Liu2, Wenting Zhang1
1Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture and Rural Affairs), Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China; Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Abstract:
Mycoplasma synoviae is a significant avian pathogen implicated in diverse clinical manifestations, including air sacculitis, infectious synovitis with arthritis, and eggshell apex abnormalities, which significantly threaten the poultry industry. To enable the rapid and accurate detection of M. synoviae, we developed a dual-mode diagnostic platform that combines recombinant enzyme-assisted amplification (RAA) with CRISPR/Cas12a-based fluorescence and lateral flow dipsticks (LFD). The Qsep100 automatic nucleic acid analysis system was used to screen for RAA primers by evaluating the amplicon size and concentration. Fluorescence monitoring revealed that the optimal reaction time for the RAA-CRISPR/Cas12a system was 28 min. After optimizing the concentrations of LbaCas12a, crRNA, and the ssDNA reporter, the assay achieved a sensitivity of 5.2 copies/µL. Cross-reactivity testing with seven common avian pathogens confirmed the high specificity of the established method for detecting M. synoviae. In the clinical validation, the method perfectly matched the results of quantitative real time polymerase chain reaction (qPCR). Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for M. synoviae among the chickens, ducks, and pigeons. In summary, we developed a rapid, accurate, and portable diagnostic platform for M. synoviae detection, which provides a valuable tool for disease prevention and control in resource-limited settings.
Insights
A new diagnostic platform rapidly and accurately detects Mycoplasma synoviae, a major poultry pathogen. This tool aids disease control in resource-limited settings.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Avian Pathology
Background:
- Mycoplasma synoviae (M. synoviae) is a significant avian pathogen causing substantial economic losses in the poultry industry.
- Current diagnostic methods for M. synoviae can be time-consuming or require specialized equipment, hindering rapid disease management.
- There is a need for a fast, accurate, and accessible diagnostic tool for M. synoviae detection.
Purpose of the Study:
- To develop and validate a novel dual-mode diagnostic platform for the rapid and accurate detection of Mycoplasma synoviae.
- To optimize the platform for sensitivity, specificity, and speed.
- To assess the platform's performance against established diagnostic methods and in field conditions.
Main Methods:
- Development of a dual-mode diagnostic platform combining Recombinant Enzyme-Assisted Amplification (RAA) with CRISPR/Cas12a technology.
- Utilized the Qsep100 system for RAA primer screening and optimization.
- Integrated fluorescence monitoring and Lateral Flow Dipsticks (LFD) for versatile detection.
- Validated the assay's sensitivity, specificity, and clinical performance against quantitative real-time PCR (qPCR).
Main Results:
- The RAA-CRISPR/Cas12a system achieved optimal reaction time at 28 minutes.
- The assay demonstrated high sensitivity (5.2 copies/µL) and specificity, with no cross-reactivity against common avian pathogens.
- Clinical validation showed perfect concordance with qPCR results.
- Epidemiological investigation identified the highest M. synoviae positivity rate in chickens.
Conclusions:
- A rapid, accurate, and portable dual-mode diagnostic platform for M. synoviae detection has been successfully developed.
- This platform offers a valuable tool for effective disease prevention and control, particularly in resource-limited settings.
- The developed assay provides a significant advancement in avian disease diagnostics.
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