Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi

The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to genera...

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Veröffentlicht in:Sensors (Basel, Switzerland) Jg. 20; H. 11; S. 3292
Hauptverfasser: Gryta, Agata, Frąc, Magdalena
Format: Journal Article
Sprache:Englisch
Veröffentlicht: Switzerland MDPI AG 09.06.2020
MDPI
Schlagworte:
Archaea - classification
Bacteria
Bacteria - classification
Biodiversity
Biosensors
capillary electrophoresis
Climate change
Deoxyribonucleic acid
DNA
Fungi
Fungi - classification
Genetic diversity
genetic fingerprinting
Genetic testing
Information sources
Land use planning
Methods
microbial genetic diversity
multiplex t-RFLP
Polymerase Chain Reaction
Polymorphism
Polymorphism, Restriction Fragment Length
Research methodology
Soil Microbiology
soil microbiome
Soil microorganisms
Taxonomy
United States > US
microbial genetic diversity
multiplex t-RFLP
genetic fingerprinting
capillary electrophoresis
soil microbiome
ISSN:1424-8220, 1424-8220
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Zusammenfassung:The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial diversity using high efficiency Multiplex PCR-Terminal Restriction Fragment Length Polymorphism (M-T-RFLP) for the simultaneous detection of bacteria, archaea and fungi. Three different primer sets were used in the reaction and the analytical conditions were optimized. Optimal analytical conditions were achieved using 0.5 µM of primer for bacteria and 1 µM for archaea and fungi, 4 ng of soil DNA template, and HaeIII restriction enzyme. Comparative tests using the proposed analytical approach and a single analysis of each microorganism group were carried out to indicate that both genetic profiles were similar. The Jaccard similarity coefficient between single and multiplexing approach ranged from 0.773 to 0.850 for bacteria and fungi, and 0.208 to 0.905 for archaea. In conclusion, the multiplexing and pooling approaches significantly reduced the costs and time required to perform the analyses, while maintaining a proper effectiveness.
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ISSN:1424-8220
1424-8220
DOI:10.3390/s20113292