ARS | Publication request: Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 25, 2006
Publication Date: December 31, 2006
Citation: Callison, S.A., Riblet, S.M., Sun, S., Ikuta, N., Hiltl, D., Leiting, V., Kleven, S.H., Suarez, D.L., Garcia, M. 2006. Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds. Avian Diseases. 50:537-544.

Interpretive Summary: Mycoplasma gallisepticum (MG) is a bacterial infection of chickens that can cause respiratory disease and loss of production in affected birds. Because respiratory disease from MG can be confused with other bacterial and viral respiratory diseases of chickens, like avian influenza on Newcastle disease virus, it is important to rapidly diagnosis the infection. This paper describes a rapid and sensitive diagnostic test for MG that was shown in both laboratory infected and naturally infected chickens to be an accurate method of diagnosis. The results were correlated with both bacterial isolation, antibody testing methods, and an alternative PCR test.

Technical Abstract: In this study, we report the development and validation of a real-time PCR assay using a Taqman labeled probe (MGLP assay) for the detection of Mycoplasma gallisepticum (M. gallisepticum). The MGLP assay was highly specific with a detection limit of 25 template copies/reaction and a quantification limit of 100 template copies/reaction. Validation of the assay was completed with 1,247 samples (palatine cleft and tracheal swabs) from M. gallisepticum positive and negative chicken flocks. The MGLP assay was compared to 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, reproducible, and allowed the quantification of template copies directly from clinical samples.