Poultry scienceNov 21, 2025

Fast detection of Mycoplasma synoviae genetic material using a two-step CRISPR-based test

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

The diagnostic platform achieved a sensitivity of 5.2 copies/µL for detecting Mycoplasma synoviae.

A dual-mode diagnostic platform was developed, combining enzyme-assisted amplification with CRISPR/Cas12a technology.
The optimal reaction time for the assay was determined to be 28 minutes.
High specificity for M. synoviae was confirmed through cross-reactivity testing with seven common avian pathogens.
Clinical validation showed perfect alignment with quantitative real-time polymerase chain reaction (qPCR) results.
Epidemiological investigation indicated that chickens had the highest positivity rate for M. synoviae compared to ducks and pigeons.

Simplified

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.

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Fast detection of Mycoplasma synoviae genetic material using a two-step CRISPR-based test

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