Development of a quantitative assay for detecting pro‐inflammatory bacteria genes directly in human stool samples by real‐time Polymerase Chain Reaction (rtPCR) (LB515)

The human intestinal microbiota plays a key role in the maintenance of health and also in the development of intestinal disorders. Numerous studies have established the definitive association between the presence of certain intestinal bacteria in the flora and the development of gastrointestinal disorders. In this work, we developed an rtPCR assay for the detection of intestinal bacteria pro‐inflammatory genes directly from human stool samples. As a test case, we chose the pks genomic island genes that are present in some strains of intestinal Escherichia coli, and have been shown to induce, megalocytosis (figure 1), DNA damage, and aberrant inflammatory response. Metagenomic DNA was extracted from anonymous stool samples and used as the template for a rtPCR reaction. The fluorophore SYBR Green I was used to visualize the real time amplification response. The linearity for the SYBR Green I response with increasing amounts of input DNA was recorded in a standard curve with a correlation value of 0.9856. The fluorescence signal was detected in all pks+ samples after 18.97 cycles for HUO33, 19.24 cycles for HUO37, 16.61 cycles for HUO38 and 24.39 cycles for HUO47. As for the pks‐ samples, three of the samples the fluorescence signal was detected at cycle 9.65 for HUO31, 10.83 cycles for HUO39 and cycle 16.02 for HUO50. The fluorescence signal detected in pks‐ sample could be attributed to contamination between sample and non‐specific amplification. However, no fluorescence signal was detected in the negative control and the blank. Work is underway for the optimization of this assay towards the screening of patient samples. Grant Funding Source : This work is supported by the National Science Foundation (NSF) grant CHE0953254 to ABO and the National Institute of Health (NIH) grant R25GM061838 (MBRS RISE Program) to RGM