Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Gryta, Agata, Frąc, Magdalena
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309186/
https://www.ncbi.nlm.nih.gov/pubmed/32527006
http://dx.doi.org/10.3390/s20113292
_version_ 1783549164390449152
author Gryta, Agata
Frąc, Magdalena
author_facet Gryta, Agata
Frąc, Magdalena
author_sort Gryta, Agata
collection PubMed
description 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.
format Online
Article
Text
id pubmed-7309186
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-73091862020-06-25 Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi Gryta, Agata Frąc, Magdalena Sensors (Basel) Article 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. MDPI 2020-06-09 /pmc/articles/PMC7309186/ /pubmed/32527006 http://dx.doi.org/10.3390/s20113292 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gryta, Agata
Frąc, Magdalena
Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi
title Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi
title_full Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi
title_fullStr Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi
title_full_unstemmed Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi
title_short Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi
title_sort methodological aspects of multiplex terminal restriction fragment length polymorphism-technique to describe the genetic diversity of soil bacteria, archaea and fungi
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309186/
https://www.ncbi.nlm.nih.gov/pubmed/32527006
http://dx.doi.org/10.3390/s20113292
work_keys_str_mv AT grytaagata methodologicalaspectsofmultiplexterminalrestrictionfragmentlengthpolymorphismtechniquetodescribethegeneticdiversityofsoilbacteriaarchaeaandfungi
AT fracmagdalena methodologicalaspectsofmultiplexterminalrestrictionfragmentlengthpolymorphismtechniquetodescribethegeneticdiversityofsoilbacteriaarchaeaandfungi