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Development of a method to extract protozoan DNA from black soil

OBJECTIVES: Microorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed by the verification of physiological characteristics and morphological classification. Isolation and purification of Amoebae from soil samples is extremely complex, labo...

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Autores principales: Yamanouchi, Kanako, Takeuchi, Masahiro, Arima, Hiroaki, Tsujiguchi, Takakiyo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324249/
https://www.ncbi.nlm.nih.gov/pubmed/30662966
http://dx.doi.org/10.1016/j.parepi.2018.e00081
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author Yamanouchi, Kanako
Takeuchi, Masahiro
Arima, Hiroaki
Tsujiguchi, Takakiyo
author_facet Yamanouchi, Kanako
Takeuchi, Masahiro
Arima, Hiroaki
Tsujiguchi, Takakiyo
author_sort Yamanouchi, Kanako
collection PubMed
description OBJECTIVES: Microorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed by the verification of physiological characteristics and morphological classification. Isolation and purification of Amoebae from soil samples is extremely complex, laborious, and time-consuming and require considerable expertise for morphological evaluation. PCR testing of soil DNA seems to be an effective means for protozoa habitat screening. In this study, we added Acanthamoeba sp. (MK strain) to soil and developed a method of extracting protozoan DNA from the soil. METHODS: Soil allophane is a known DNA adsorbing substance that inhibits the PCR reaction. After comparing the soil properties and allophane contents of 7 soil samples, we attempted to combine multiple cell disruption and DNA purification methods to design an optimal soil DNA extraction method that can be used for downstream PCR analysis. RESULTS: We compared five different crushing/refining methods. Amplification of the gene was confirmed by Acanthamoeba specific PCR in protocol V where the concentration of Acanthamoeba in soil (1.0 × 10(2)/g) was the detection limit of PCR. CONCLUSION: The soil DNA extraction method following protocol V allows DNA amplification of protozoa, including Amoeba, which is difficult to cultivate, thus simplifying the investigation of protozoa habitats and genetic analyses.
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spelling pubmed-63242492019-01-18 Development of a method to extract protozoan DNA from black soil Yamanouchi, Kanako Takeuchi, Masahiro Arima, Hiroaki Tsujiguchi, Takakiyo Parasite Epidemiol Control Article OBJECTIVES: Microorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed by the verification of physiological characteristics and morphological classification. Isolation and purification of Amoebae from soil samples is extremely complex, laborious, and time-consuming and require considerable expertise for morphological evaluation. PCR testing of soil DNA seems to be an effective means for protozoa habitat screening. In this study, we added Acanthamoeba sp. (MK strain) to soil and developed a method of extracting protozoan DNA from the soil. METHODS: Soil allophane is a known DNA adsorbing substance that inhibits the PCR reaction. After comparing the soil properties and allophane contents of 7 soil samples, we attempted to combine multiple cell disruption and DNA purification methods to design an optimal soil DNA extraction method that can be used for downstream PCR analysis. RESULTS: We compared five different crushing/refining methods. Amplification of the gene was confirmed by Acanthamoeba specific PCR in protocol V where the concentration of Acanthamoeba in soil (1.0 × 10(2)/g) was the detection limit of PCR. CONCLUSION: The soil DNA extraction method following protocol V allows DNA amplification of protozoa, including Amoeba, which is difficult to cultivate, thus simplifying the investigation of protozoa habitats and genetic analyses. Elsevier 2018-12-29 /pmc/articles/PMC6324249/ /pubmed/30662966 http://dx.doi.org/10.1016/j.parepi.2018.e00081 Text en © 2018 Published by Elsevier Ltd on behalf of World Federation of Parasitologists. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Yamanouchi, Kanako
Takeuchi, Masahiro
Arima, Hiroaki
Tsujiguchi, Takakiyo
Development of a method to extract protozoan DNA from black soil
title Development of a method to extract protozoan DNA from black soil
title_full Development of a method to extract protozoan DNA from black soil
title_fullStr Development of a method to extract protozoan DNA from black soil
title_full_unstemmed Development of a method to extract protozoan DNA from black soil
title_short Development of a method to extract protozoan DNA from black soil
title_sort development of a method to extract protozoan dna from black soil
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324249/
https://www.ncbi.nlm.nih.gov/pubmed/30662966
http://dx.doi.org/10.1016/j.parepi.2018.e00081
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