Detection of Mycoplasma synoviae by polymerase chain reaction

A Mycoplasma synoviae (Ms) species-specific recombinant subclone, pMS156-20, of approximately 1.1 kbp was partially sequenced. Based on the sequence data, a pair of 25 base primers were synthesized to develop a Ms polymerase chain reaction (Afs-PCR). The primers amplified target DNA of approximately 1.1 kbp and had excellent specificity over a range of annealing temperature from 60 degrees C to 68 degrees C. The primers amplified 100 pg of 26 strains or isolates of Ms, but not three strains of M. gallisepticum (S6, K810, A5969), 15 other avian Mycoplasma species, and pUC8 plasmid. The minimum amount of target DNA detected by Ms-PCR was 10 fg, which was 100,000 times more sensitive than the dot blot methodology using Ms recombinant DNA probes. The specificity of Ms-PCR product detected by gel electrophoresis was confirmed by Southern blot hybridization using an internal probe derived from pMSl56-20.