The method of DNA extraction from soil samples

Abstract

Introduction. Even in the modern urban environment humans are in constant direct and indirect contact with soil. This leads to the spread of a wide range of soil-transmitted human and animal pathogens. Therefore, the development of fast and inexpensive methods of analysis and monitoring of these pathogenic objects is of great importance. PCR method is widely applied in laboratory practice and is able to detect even the uncultivated types of pathogens. The aim of the study was to optimize the method of DNA isolation from soil, making it suitable for PCR-assay. Materials and methods. DNA was isolated from the samples of surface layer of forest soil rich in humus, using lab-shelf chemicals and/or commercial kit. RT-PCR-test was performed using universal bacterial primers. Results. We have analyzed various combinations of four extraction methods and three pre- and post-treatment methods. DNA was efficiently extracted by all methods, however, without additional purification stages it was unsuitable for PCR. The calcium salts treatment ws demonstrated to be necessary for removal of PCR inhibitors, presumably humic acids. Two DNA isolation methods were developed. Both methods use incubation with CaCO3 suspension followed by cetrimonium bromide lysis. More sensitive and unexpensive method uses CaCl2 as an additional purification stage. The less sensitive but more reproducible method included DNA isolation on Qiagen DNA (Qiagen) columns. Limitations. When working out the technique of DNA isolation for PCR analysis, samples of the only sod-podzolic soil were studied. Therefore, the technique can be applied only for this type of soil. Conclusion. Both methods optimized in this study can be used for evaluation of soil samples for the presence of pathogens by PCR.