EFFECT OF EXTRACTION METHOD AND DNA QUALITY ON THE RELIABILITY OF MOLECULAR DETECTION OF BRADYRHIZOBIUM JAPONICUM IN SOYBEAN RHIZOSPHEREDOI:10.30825/5.EJPAU.175.2019.22.2

摘要

Several protocols of DNA extraction from the soil with commercial kits and CTAB based method were tested to develop relatively easy procedure of reliable molecular detection of B. japonicum introduced with coated soybean seeds. Method of DNA extraction clearly influences the quality and quantity of DNA and thus directly affects the results of PCR (Polymerase Chain Reaction) and qPCR (quantitative Polymerase Chain Reaction) assays and the possibility of bacteria identification. Obtaining reliable results in PCR does not guarantee the same results in qPCR with SYBR Green. The use of DNA extraction commercial kits and successful DNA extraction may in some cases lead to inability to detect bacteria, although it is present in the sample. To avoid such problems, the extraction methods should be selected and, if necessary, modified individually for different soil samples. In this study, DNA extraction with Genomic MINI AX Soil Spin showed to be the most appropriate method for obtaining reliable results in both PCR and real-time PCR. However, for real-time PCR, strong dilution of DNA and the addition of reagents that relieve inhibition of amplification were necessary.