Isolation and detection of Mycoplasma gallisepticum by polymerase chain reaction and restriction fragment length polymorphism

ABSTRACT

Mycoplasmas were isolated from tracheal and air sac samples of the suspected flocks to have mycoplasmosis and cultured on Frey's medium. Forward and reverse primers were selected on the basis of known sequences of Mycoplasma gallisepticum [MG] and Mycoplasma synoviae [MS] 16S rRNA genes. These primers successfully amplified a 780 bp fragment of the target DNA in MG. Restriction fragment length polymorphism [RFLP] by three restriction enzymes [REs], Hpal, Hpa II, and Mbol was performed for each PCR product. According to the RFLP results, 55 samples were detected as MG; no MS was detected. The PCR results were confirmed by sequencing of a selected amplicons. Results showed that PCR and RFLP are rapid and useful for diagnosis of both cultured as well as field samples of suspected flocks to have infection with MG. In addition, PCR could be performed with at least 100 CFU of MG per each PCR reaction