Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.

Copyright © Wydawnictwo Akademii Rolniczej we Wroclawiu, ISSN 1505-0297

Baturo-Cieśniewska A. , Loddi G. , Prusiński J. , Łukanowski A. 2019. EFFECT OF EXTRACTION METHOD AND DNA QUALITY ON THE RELIABILITY OF MOLECULAR DETECTION OF BRADYRHIZOBIUM JAPONICUM IN SOYBEAN RHIZOSPHERE
DOI:10.30825/5.ejpau.175.2019.22.2 , EJPAU 22(2), #05.
Available Online: http://www.ejpau.media.pl/volume22/issue2/abs-05.html

ABSTRACT

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.

Key words: Bradyrhizobium japonicum, DNA extraction, PCR inhibitors, real-time PCR, soil.

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Anna Baturo-Cieśniewska
Laboratory of Phytopathology and Molecular Mycology, Department of Biology and Plant Pathology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
Kordeckiego St. 20
85-225 Bydgoszcz
Poland
email: baturo-a@utp.edu.pl

Giuseppe Loddi
Consortium UNO, Faculty of Biology and Pharmacy, University of Cagliari, Oristano, Italy
Carmine St.
09170 Oristano
Italy

Janusz Prusiński
Department of Agrotechnology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
Kordeckiego St. 20
85-225 Bydgoszcz
Poland
email: janusz.prusinski@utp.edu.pl

Aleksander Łukanowski
Laboratory of Phytopathology and Molecular Mycology, Department of Biology and Plant Pathology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
Kordeckiego St. 20
85-225 Bydgoszcz
Poland

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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