An improved real-time qPCR technique for quantification of intestinal bacteria in human fecal samples

Abstract

The easy, cost-effective and rapid techniques for investigating the role of gut microbiota have become imperative owing to their importance in various diseases such as colon cancer and inflammatory bowel disease. Here we developed an absolute real-time qPCR technique for quantification of intestinal bacteria in human fecal samples for further investigation of gut microbiota composition in patients. At first, regarding bacterial species present in human fecal samples, species were cultured in anaerobic culture media and incubated at 37Cͦ in an anaerobic chamber. The number of CFU was counted. Eight fold serial dilutions of the bacterial suspension were prepared. To generate a standard curve for bacterial species in real-time PCR, DNA was extracted from different serial dilutions. Subsequently, DNA from human fecal samples was extracted. Now bacterial strains were applied as standard curve in absolute q PCR procedure for quantification of bacterial population in test samples. Based on absolute real-time analysis, intestinal bacteria were precisely quantified. The specificity of the primer pairs was checked by conventional PCR and real-time PCR regarding dissociation curve analysis. The amplification was linear in the range of 101-108. Dissociation curves had correlation coefficient values between 0.98 and 101/888. The efficiencies were between 95/073 and 99/804%. The absolute real-time PCR technique described here may be used for evaluating intestinal bacteria regarding diseases prevention.