Development of a method to extract protozoan DNA from black soil.

摘要: Abstract Objectives Microorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed the verification of physiological characteristics morphological classification. Isolation purification Amoebae from soil is extremely complex, laborious, time-consuming require considerable expertise for evaluation. PCR testing DNA seems to be an effective means protozoa habitat screening. In this study, we added Acanthamoeba sp. (MK strain) developed a method extracting protozoan soil. Methods Soil allophane known adsorbing substance that inhibits reaction. After comparing properties contents 7 samples, attempted combine multiple cell disruption methods design optimal extraction can used downstream analysis. Results We compared five different crushing/refining methods. Amplification gene was confirmed specific protocol V where concentration (1.0 × 102/g) detection limit PCR. Conclusion The following allows amplification protozoa, including Amoeba, which difficult cultivate, thus simplifying investigation habitats genetic analyses.