OBJECTIVE To establish multiple PCR(M-PCR) assay for simultaneous detection of Treponema pallidum(TP) and herpes simplex virus(HSV),Haemophilus ducreyi(HD).METHODS According to specific gene sequence of the three agents:HSV DNA polymerse gene,TP tpp47 gene,HD 6s rRNA gene;three sets of specific primers were designed and a multiple PCR assay was developed to detect 3 agents in one test.RESULTS The target DNA of 3 agents were specifically amplified by their specific primers,the appropriate size of four DNA fragments was 202bp,252bp,152bp for TP,HSV and HD,respectively.The DNA of other several common microbes of genital tract and human genome could not be amplified by three sets of primers.CONCLUSIONS Primary study indicated that the M-PCR assay can simultaneously,specifically and rapidly detect out HSV,TP and HD.