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We developed a one-step method based on multiplex-overlapping polymerase chain reaction (mPCR) for species differentiation of Pediococcus acidilactici and P. pentosaceus as well as simultaneous detection of pediocin (ped) gene. The mPCR was performed in a single reaction tube with six primers consisting of four primers for identification of P. acidilactici and P. pentosaceus and two primers for pediocin gene amplification. We distinguished both species from nine other species of pediococci as well as several other lactic acid bacteria (LAB). The identification performed by this mPCR was confirmed by partial 16S rDNA sequencing of selected pediococcal and LAB isolates. This multiplex-overlapping PCR is a simple, rapid determination and reliable method for differentiation of both pediococcal species as well as determination of the ability to produce pediocin in a single PCR reaction. [ABSTRACT FROM AUTHOR]