Method for selective enrichment and isolation of microbial and optional additionally of viral nucleic acids

摘要

Selective enrichment and/or isolation of microbial nucleic acids from samples, comprises: (i) separating the liquid components from the solid components of the sample; (ii) selective lysis of the eukaryotic cells contained in the solid sample components without simultaneous lysis of microbial cells; (iii) separating the solid components comprising the microbial cells from the liquid component; (iv) re-suspending the solid obtained in the step (iii); (v) carrying out lysis of microbial cells; (vi) cleaning and or concentrating the nucleic acids from liquid component obtained in the step (i). Selective enrichment and/or isolation of microbial nucleic acids from samples comprising a mixture of microbial cells, higher eukaryotic cells and free circulating nucleic acids, preferably extracellular microbial nucleic acids contained in a liquid, comprises: (i) separating the liquid components of the sample comprising those substances dissolved in the liquid component, e.g. the free circulating nucleic acids, preferably the extracellular nucleic acids, from the solid components of the sample, or from the cellular components; (ii) selective lysis of the eukaryotic cells contained in the solid sample components without simultaneous lysis of microbial cells; (iii) separating the solid components comprising the microbial cells from the liquid component, which comprises those substances dissolved in the liquid component, and the lysate obtained in the step (ii); (iv) re-suspending the solid obtained in the step (iii); (v) carrying out lysis of the microbial cells, which are present in the suspension obtained in the step (iv); and (vi) cleaning and or concentrating the freely circulating, preferably the extra-cellular microbial, nucleic acids from the liquid component obtained in the step (i) and/or intracellular microbial nucleic acids from the lysate obtained in the step (v). An independent claim is also included for a kit for selective enrichment and/or isolation of intracellular microbial and freely circulating, preferably extracellular microbial nucleic acids and optionally additional viral nucleic acids from samples, which comprises a mixture of microbial higher eukaryotic cells and free circulating nucleic acids, preferably extracellular microbial nucleic acids, and optionally additional viruses in a liquid, comprising (iia) a first buffer comprising 0.2-4 M chaotrope, preferably comprising guanidinium, 0.5-10% (w/v) surfactant, preferably a nonionic surfactant and 2-100 mM buffer substance, preferably at a pH of 8, (iib) at least one second buffer, comprising 5-50 mM buffer substance, 0.5-10 mM complexing reagent, 0.2-10% (w/v) surfactant and optionally 0.1-3% (v/v) foam inhibitor at a pH of 8, and/or comprising 3-10 M chaotrope, 10-40% (w/v) surfactant and 40-200 mM buffer substance at a pH 8, (iic) glass beads with 400-600 mu m diameter, preferably 500 mu m.