Simple and cost-effective protocol for bacterial DNA extraction from stool samples, suitable for PCR and qPCR

OUSSET J; VENTURINO A; JAUREGUIBERRY MS

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

In recent years we have witnessed a paradigm shift regarding the microbiome and its role in health and disease. Our distal gastrointestinal tract hosts for over a trillion bacteria of several species and dysbiosis (imbalance of microbial community composition and functionality) has been shown to underlie a wide range of diseases, including not only gastroenterologic issues, but also immune, respiratory, metabolic, hepatic, cardiovascular and even neurologic disorders such autism, Parkinson?s disease, anxiety and depression. Thus, microbiome characterization studies have become a topic of great interest in current research, many of which require bacterial DNA extraction from stool samples. Because of the highly complex composition of feces, consistent extraction of high-quality DNA from fecal samples is challenging due to the presence of inhibitors (i.e., bile salts, lipids, urate and complex polysaccharides) that might be co-extracted with DNA, and could affect downstream techniques such as PCR, dramatically reducing sensitivity and amplification efficiency. Several methods have been proposed in order to extract DNA from feces that are, in some cases, laborious and combine multiple purification steps. DNA extraction commercial kits are a very popular choice. Although efficient and convenient, they are comparatively expensive and sometimes, like other methods, fail with samples from herbivorous species. They also include reagents of unknown composition (e.g. lysis buffer, inhibitors absorber), and silica gel membrane to further remove inhibitors from the DNA solution. Considering complex polysaccharides from diet are major PCR inhibitors, we adapted and modified a CTAB-PVP based procedure for plant RNA extraction, and developed a cost effective, simple workflow that renders quality, non-fragmented DNA suitable for PCR and RT-PCR from feces. Moreover, our method replaces expensive enzymes (for breaking down bacterial cell wall) such as lysozyme or proteinase-K with liquid nitrogen rupture, and the optional use of silica columns for further purification.