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Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay
Hung-Chih Kuo1, Dan-Yuan Lo1, Chiou-Lin Chen1
1Yunlin-Chiayi-Tainan of Animal Disease Diagnostic Center, Department of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan.
Abstract:
Mycoplasma synoviae (MS), causing respiratory diseases, arthritis, and eggshell apex abnormalities in avian species, is an important pathogen in the poultry industry. Implementation of a biosecurity plan is important in MS infection management. Working on a field-deployable POCKIT™ device, an insulated isothermal polymerase chain reaction (iiPCR) assay has a potential for timely MS detection on the farm. The MS iiPCR assay had limit of detection 95% of about 9 genome equivalents by testing serial dilutions of a standard DNA. The detection endpoint of the assay for detection of MS genomic DNA was comparable to a reference real-time PCR. The assay did not crossreact with other important avian pathogens, including avian reovirus, Mycoplasma gallisepticum, Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Salmonella Pullorum. When 92 synovial fluid and respiratory tract swab samples collected from chickens, turkeys, and geese suspected of MS infection were tested, the clinical performance of the MS iiPCR had 97.8% agreement (Cohen's kappa value, 0.95) with that of the reference real-time PCR. In conclusion, the MS iiPCR/POCKIT™ system, working with field-deployable manual or automatic nucleic acid extraction methods, has potential to serve as a rapid and sensitive on-site tool to facilitate timely detection of MS.
Insights
A new insulated isothermal polymerase chain reaction (iiPCR) assay on the POCKIT™ device offers rapid, on-farm detection of Mycoplasma synoviae (MS) in poultry. This sensitive tool aids in timely management of MS infections, improving flock health.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Avian Health
Background:
- Mycoplasma synoviae (MS) is a significant poultry pathogen causing respiratory illness, arthritis, and eggshell defects.
- Effective management of MS relies on prompt and accurate on-farm diagnostics.
- Current diagnostic methods may not always be suitable for rapid field deployment.
Purpose of the Study:
- To evaluate a novel insulated isothermal polymerase chain reaction (iiPCR) assay for the detection of Mycoplasma synoviae (MS).
- To assess the performance of the MS iiPCR assay on the field-deployable POCKIT™ device for on-site diagnostics.
- To determine the clinical utility of the MS iiPCR assay in avian species suspected of MS infection.
Main Methods:
- The MS iiPCR assay was developed and validated using serial dilutions of MS genomic DNA to determine its limit of detection.
- Assay specificity was confirmed by testing against a panel of common avian pathogens.
- Clinical performance was evaluated by comparing results from the MS iiPCR assay with a reference real-time PCR using field samples (synovial fluid and respiratory swabs).
Main Results:
- The MS iiPCR assay demonstrated a high sensitivity with a limit of detection of approximately 9 genome equivalents.
- The assay showed excellent specificity, with no cross-reactivity observed with other tested avian pathogens.
- Clinical evaluation revealed a high agreement (97.8%, Cohen's kappa = 0.95) between the MS iiPCR assay and the reference real-time PCR.
Conclusions:
- The MS iiPCR assay, when used with the POCKIT™ system and field-deployable nucleic acid extraction, is a rapid, sensitive, and specific tool for on-site MS detection.
- This diagnostic system has the potential to significantly improve the timely management of Mycoplasma synoviae in the poultry industry.
- The assay facilitates early disease detection, enabling prompt implementation of control measures to mitigate economic losses associated with MS.
