A new pentaplex-nested PCR to detect five pathogenic bacteria in free living amoebae

► We have developed a pentaplex PCR assay for detection of five pathogenic bacteria. ► The assay is suitable for detection in water sources or inside free-living amoeba. ► Sensitivity of the procedure is similar to those reported for q-RT-PCR assays. ► Validation of the assay shows predominance of Mycobacterium in samples from Aragon.