Development and Optimization of Quantitative Real Time PCR for the Detection of Mycoplasma Synoviae Infections

Introduction: Mycoplasma synoviae (MS) is a major pathogen of chickens and turkeys causing upper respiratory infection and synovitis in chickens and turkeys. Additionally, MS infection in turkeys has been associated with increased condemnation at processing due to airsacculitis, as well as with breast blisters and green discoloration of liver, which results in slower processing and further economic loss. Because of the high value of individual breeder hens and because of their ability to infect progeny by vertical transmission, economic losses are potentially most severe when MS infections occur in breeder flocks. Therefore a reasonable strategy for control of MS is to establish MS-free breeder flocks. Diagnosis of MS infections is often carried out by serological procedures but non specific reactions or insensitivity of the serological detection of MS infection often limits the effectiveness of these procedures. Isolation of the organism is also used but may be unsuccessful due to bacterial contamination, mixed mycoplasma infections or other reasons. Successful isolation may require 1-3 weeks. Species specific PCR tests are used based on 16s rRNA and vlhA genes. The major advantages of Real-Time PCR over standard PCR assay are the one tube and one time handling with reading of the reaction during the assay and less time needed to perform the test.

Objective: To develop and optimize real time PCR assay for quantitative detection of Mycoplasma synoviae infections from clinical samples.

Methodology: M. synoviae strains/isolates from chicken and turkey will be procured for standardization of the real time PCR assay. Clinical samples (trachea, air sac, synovial fluid) from affected chicken/turkey will be collected. Nucleic acids will be extracted directly from different M. synoviae strains/isolates and clinical samples and will be subjected to real-time PCR. Primer and fluorescent tagged probe set used in this investigation will be developed and characterized. The amplification of DNA fragment from the suspect samples detected by monitoring the increase in fluorescence from the dye labeled MS-specific probe will be investigated