A simple and practical method for simultaneous detection of Ureaplasma urealyticum (U. urealyticum), Ureaplasma parvum (U. parvum), Mycoplasma hominis (M. hominis) and Mycoplasma genitalium (M. genitalium) by fluorescence polarization (FP) assay has been developed. DNA of the 16S rRNA gene of U. urealyticum, U. parvum, M. genitalium and M. hominis was amplified using a forward primer and two reverse primers in an asymmetric PCR. Four probes labeled with different fluorophores were specific for U. urealyticum, U. parvum, M. hominis and M. genitalium, respectively. The probes hybridized with their target PCR products, and the hybridization increased the fluorescence polarization (FP) values. The pathogens were identified by the increased FP values. DNA extracted from 757 urethral samples was detected by the FP assay and the sequence method. The FP assay could detect co-infection more effectively than the sequence method did. The method presented in this study was expected to be extremely useful in clinic.
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