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Foot-and-mouth disease (FMD) is an extremely contagious and economically important viral disease affecting livestock. Rapid and precise diagnosis of FMD is of critical importance for efficient control and surveillance strategies of the disease. In this study, one-step real-time reverse transcription-polymerase chain reaction (RTqPCR) assays were developed using newly designed primers/probe sets in the conserved regions within the VP1 coding sequence for specific detection of FMDV serotypes SAT 2 and O with their different lineage circulating in Egypt. The assays were validated for efficacy to detect different lineages of these endemic FMDV serotypes in Egypt; the detection limit was 10 genomic copies for serotype SAT 2 and one genomic copy for serotype O, with no cross-reactivity observed. These findings were confirmed by the specific and sensitive detection of FMDV in clinical samples obtained from different regions in Egypt and representing a range of subtypes within the SAT 2 and O serotypes. The results illustrated the potential of tailored RT-qPCR methods for the rapid detection and serotyping of FMDV belonging to different lineages of serotypes SAT 2 and O circulating in Egypt with high sensitivity and specificity. The developed assays could be easily deployed for routine surveillance and hence improving the disease control measures.