Development of a method to extract protozoan DNA from black soil

Abstract

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 × 102/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. Keywords: Soil, DNA, Protozoan, PCR inhibitor, Allophane, Epidemiological survey