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The gene coding for antimicrobial peptides produced by probiotic Lactobacillus reuteriKUB-AC5 located on the cloned DNA fragment I-C46 containing 2 open reading frames I-C46-F2.1 and I-C46-F2.2 were designed for strain-specific primers P1 and P2, respectively, and assessed by real-time quantitative polymerase chain reaction. According to the obtained results, primer P1 has limited strain specificity. Primer P2 exhibited high efficacy and specificity at annealing temperature of 70°C while P1 annealed at 57°C causing nonspecific bands. Hence, P2 was selected for quantitative polymerase chain reaction assay by isothermal annealing and extension reaction at high temperature of 70°C resulting in linearity for its DNA sequences ranging from 102to 107target copy numbers per assay, and displaying a detection limit of 6.17 log cfu/g of cecal digesta. Using spike testing, this system was able to detect 7.88 ± 0.06 to 11.78 ± 0.06 log copy number/g of digesta, higher than cultivation assay at about 1 log cfu/g, with good correlation of 0.99. These results suggested possible detection of strain KUB-AC5 in the gastrointestinal tract of chicken to evaluate the efficacy and persistence of a probiotic strain which requires correct inclusion rates in the feed.