Objective To develop a PCR-mverse dot blot hybridization(RDB)assay to rapidly detect pathogenic mycoplasmas in genitourinary tract.Methods Universal primers were designed and applied to amplify the 16S rRNA gone of ureaplasma parvum(Up),ureaplasma urealyticum(Uu),Mycoplasma genitalium(Mg),Mycoplasma hominis(Mh)by using nestcd PCR.Specific nucleotide probes of Up,Uu,Mg and Mh Were constructed and immobilized onto nylon membranes.PCR products were denatured and hybridized、with specific oligonucleofide probes on nylon membrane.The sensitivity and specificity of the PCR-RDB assay were evaluated based.on the hybddizafion results.Also,PCR-RDB Was utilized to detect pathogenic mycoplasmas from 60 clinical samples.Results The four probes selectively hybridized with the PCR product of corresponding mycoplasmas,and no cross hybridization was observed.The detection limit of PCR-RDB Was one colony forming unit(CFU)of mycoplasma.Out of the 60 clinical samples、19were positive for mycoplasm,Mixed infections were found in three samples,including two coinfected with Up and Uu and one with Uu and Mg.Conclusion PCR-RDB is a rapid,specific and sensitive approach to the identification of pathogenic mycoplasmas in urogenital tract.