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Background and objectives: A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an indication on anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. Materials and Methods: The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Results: Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method. Conclusion: The results showed that the semi-quantitative RT-PCR method has the clear potential to be useful as a powerful tool in early detection of anastomotic leakage.