M53 stardand strains were detected using one-step method amplipifiction and southern boltting a pair of oligonuoleotide primers and probe, were designed, synthesized and purified a specific, sensitiv and rapid detected meothod on agarose gel eletrophoresis products showed the 710bp specific band of M53 genomice DNAs. of 50 subjects were detected from SD rats, the results suggest that test-rate of PCR was higher than that of isolates cultivate method. The amplified products were confirmed by southern boltting, using alkaline phophatase (AP) labeled oligonuoeleotide probe and the specificity DNAs sequence by southern blotting onto Nylon Hybond-N membranes, and the negtive cotrans were no signals. By PCR specific detection, as little as 10pg of Mycoplasma Pulmonis DNAs yielded positive results. These results indicated that one-step PCR assay was rapid, specific, sensitive, and suitable for the detection of rats and mice.