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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2018
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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. |
format | Online Article Text |
id | pubmed-6324249 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
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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