The present invention provides methods and reagents for the detection and identification of four causative agents of genital alterations, herpes simplex virus (HSV) types 1 and 2, Treponema pallidum, and Haemophilus ducreyi. The methods use sequence-specific primers which enable the simultaneous polymerase chain reaction amplification of genomic nucleic acid sequences from HSV types 1 and 2, T. pallidum, and H. ducreyi. Following amplification, sequence-specific oligonucleotide probes are used to detect and distinguish HSV type 1 and 2, T. pallidum, and H. ducreyi nucleic acid.
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