Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds.
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.
Callison
S A
SA
Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, The University of Georgia, 953 College Station Road, Athens, GA 30602, USA.
Riblet
S M
SM
Sun
S
S
Ikuta
N
N
Hilt
D
D
Leiting
V
V
Kleven
S H
SH
Suarez
D L
DL
García
M
M
D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D045686:Mycoplasma gallisepticum / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial