scope:

This standard describes the quality requirements for the analysis of agricultural soils in order to determine the direct or indirect effects of the release or cultivation of GMOs on the structure and function or functional potentials of microbial communities. Given an optimum extraction and detection system, the nucleic acids can be used to detect transgenic DNA (recombinant genes) introduced into soil through GMOs or their residues by qualitative or quantitative means (for this purpose see VDI 4330 Part 7).

The methods described relate to the cultivationindependent analysis of microbial communities. The aim is to isolate the nucleic acids with the highest possible molecular weight from different soils using suitable extraction methods and to purify them by removing any accompanying substances, which could impede subsequently applied molecular detection methods. Extraction methods must be adapted to the specific characteristics of the soil if necessary. It would not be particularly helpful in the context of this standard to define one standard protocol for the extraction of DNA and/or RNA from soil, since the yield of extracted nucleic acids, their molecular weight and the concentration of co-extracted soil components often vary depending on the soil characteristics. Therefore this standard describes the extraction and purification steps in a modular structure and, in the Annex, gives examples of protocols to extract DNA/RNA from soils. It is further intended to give specific advice when dealing with critical extraction and purification parameters.

Direct extraction methods are recommended in this standard for extraction of nucleic acids from soil matrices. This approach automatically includes to a certain extend the extraction of microbial cellular DNA and RNA as well as extracellular cellfree DNA, including those originating from plant debris. It should be noted that cell-free DNA has a high affinity to be sorbed by particle-active surfaces in soil, particularly clay minerals and extraction of DNA from such compartments may not be quantitative.

Cell extraction is recommended to investigate microbial communities from the rhizosphere (see also introduction) on the basis of their SSU rRNA genes. The microbial fraction is first obtained through centrifugation and subsequently used for nucleic acid extraction. This approach largely prevents contamination with plant plastids or mitochondria, which also contain SSU rRNA genes. Other bacterial genes from the rhizosphere can also be detected from directly extracted DNA.