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1 December 2006 Development and Validation of a Real-Time Taqman® Polymerase Chain Reaction Assay for the Detection of Mycoplasma gallisepticum in Naturally Infected Birds
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Abstract

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman®-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M. gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.

S. A. Callison, S. M. Riblet, S. Sun, N. Ikuta, D. Hilt, V. Leiting, S. H. Kleven, D. L. Suarez, and M. García "Development and Validation of a Real-Time Taqman® Polymerase Chain Reaction Assay for the Detection of Mycoplasma gallisepticum in Naturally Infected Birds," Avian Diseases 50(4), 537-544, (1 December 2006). https://doi.org/10.1637/7639-050106R.1
Received: 1 May 2006; Accepted: 1 June 2006; Published: 1 December 2006
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