• Journal of Microbiology
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• v.34 no.3
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• pp.229-235
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• 1996

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

• Journal of Soil and Groundwater Environment
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• v.14 no.3
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• pp.57-67
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• 2009

In soil and sediment environment, microorganisms play major roles in biochemical cycles of ecological significant elements. Because of its ecological significance, microbial diversity and community structure information are useful as indexes for assessing the quality of subsurface ecological environment and bioremediation. To achieve more accurate assessment, it is requested to gain sufficient yield and purity of DNA extracted from various soil and sediment samples. Although there have been a large number of basic researches regarding soil and sediment DNA extraction methods, little guideline information is given in literature when choosing optimal DNA extraction methods for various purposes such as environmental ecology impact assessment and bioremediation capability evaluation. In this study, we performed a thorough literature review to compare the characteristics of the current DNA extraction methods from soil and sediment samples, and discussed about considerations when selecting and applying DNA extraction methods for environmental impact assessment and bioremediation capability evaluation. This review suggested that one approach is not enough to gain the suitable quantity and yield of DNA for assessing microbial diversity, community structure and population dynamics, and that a careful attention has to be paid for selecting an optimal method for individual environmental purpose.

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• 보존과학연구
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• s.29
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• pp.137-148
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• 2008

Analyses of ancient DNA (aDNA) from archaeological and historical skeletal material are characterized by low quality. Many soil contaminants such as humic acid, fulvic acid, and bone collagen are often co-extracted with aDNA and inhibit amplification by polymerase chain reaction (PCR). In this study, we compared with two methods of DNA extraction by phenolchloroform extraction and silica-bead extraction. In addition, we applied new protocol, ultra sonication based silica-bead extraction method to extract aDNA from some ancient human skeletal remains. This method was more effective by both mitochondrial DNA (mtDNA) and amelogenin gene amplification.

• Journal of Applied Biological Chemistry
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• v.56 no.2
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• pp.95-100
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• 2013

Soil microbes are important integral components of soil ecosystem which have significant and diverse role in organic matter decomposition, nitrogen cycling, and nitrogen fixation. In this study an effective denaturing gradient gel electrophoresis (DGGE) method was employed for paddy soil microbial diversity survey. For optimum paddy soil microbial DNA extraction, different methods such as Lysis buffer, skim milk bead, sodium phosphate buffer, Epicentre Soil Master DNA extraction kit (Epicentre, USA) and Mo Bio Power Soil DNA kit (MO BIO, USA) methods were utilized. Among all the method, using Mo Bio Power Soil kit was most effective. DGGE analysis of Bacteria was carried out at 6% polyacylamide gel and 45-60% denaturing gradient in the optimal conditions. Whereas DGGE analysis of fungi was done at 6% polyacrylamide gel and 45-80% denaturing gradient in the optimal conditions. By applying the above assay, it was found that variation within the microbial community of paddy soil occurs by a factor of time. DGGE assay used in this study through for a variety of soil microbial analysis suggests the potential use of this method.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.100-107
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• 2000

In a recently developed strategy for in-situ treatment of polychlorinated biphenyls (PCB), bioaugmentation was used in conjunction with a surfactant, sorbitan trioleate, as a carbon source for the degrader bacteria, along with the monoterpene, carvone, and salicylic acid as inducing substrates. Two bacteria were used for soil inoculants, including Arthrobacter sp. st. B1B and Ralstonia eutrophus H850. This methodology achieved 60% degradation of PCBs in Aroclor 1242 after 18 weeks in soils receiving 34 repeated applications of the degrader bacteria. However, an obvious limitation was the requirement for soil mixing after every soil inoculation. In the research reported here, bioaugmentation and biostimulation treatment strategies were modified by using the earthworm, Pheretima hawayana, as a vector for dispersal and mixing of surface-applied PCB-degrading bacteria and soil chemical amendments. Changes in microbial biomass and microbial community structure due to earthworm effects were examined using DNA extraction and PCR-DGGE of 16S rDNA. Results showed that earthworms effectively promoted biodegradation of PCBs in bioaugmented soils to the same extent previously achieved using physical soil mixing, and had a lesser, but significant effect in promoting PCB biodegradation in biostimulated soils treated with carvone and salicylic acid. The effects of earthworms were speculated to involve many interacting factors including increased bacterial transport to lower soil depths, improved soil aeration, and enhanced microbial activity and diversity.

• The Plant Pathology Journal
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• v.33 no.2
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• pp.184-192
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• 2017

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at $63^{\circ}C$ for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was $10^{-2}J2/0.5g$ of soil, which was 10 times more sensitive than conventional PCR ($10^{-1}J2/0.5g$ of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.170-189
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• 2021

Environmental DNA (eDNA) is a genetic material derived from organisms in various environments (water, soil, and air). eDNA has many advantages, such as high sensitivity, short investigation time, investigation safety, and accurate species identification. For this reason, it is used in various fields, such as biological monitoring and searching for harmful and endangered organisms. To collect eDNA from a freshwater ecosystem, it is necessary to consider the target organism and gene and a wide variety of items, such as on-site filtration and eDNA preservation methods. In particular, the method of collecting eDNA from the environment is directly related to the eDNA concentration, and when collecting eDNA using an appropriate collection method, accurate (good quality) analysis results can be obtained. In addition, in preserving and extracting eDNA collected from the freshwater ecosystem, when an accurate method is used, the concentration of eDNA distributed in the field can be accurately analyzed. Therefore, for researchers at the initial stage of eDNA research, the eDNA technology poses a difficult barrier to overcome. Thus, basic knowledge of eDNA surveys is necessary. In this study, we introduced sampling of eDNA and transport of sampled eDNA in aquatic ecosystems and extraction methods for eDNA in the laboratory. In addition, we introduced simpler and more efficient eDNA collection tools. On this basis, we hope that the eDNA technique could be more widely used to study aquatic ecosystems and help researchers who are starting to use the eDNA technique.

• Mycobiology
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• v.38 no.1
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• pp.33-38
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• 2010

We have successfully applied the nested PCR to detect Cylindrocarpon destructans, a major pathogen causing root rot disease from ginseng seedlings in our former study. The PCR assay, in this study, was used to detect the pathogen from soils. The nested PCR using internal transcribed spacer (ITS) 1, 4 primer set and Dest 1, 4 primer set maintained the specificity in soils containing various microorganisms. For a soil DNA extraction method targeting chlamydospores, when several cell wall disrupting methods were tested, the combination of lyophilization and grinding with glass beads, which broke almost all the chlamydospores, was the strongest. The DNA extraction method which was completed based on the above was simple and time-saving because of exclusion of unnecessary stages, and efficient to apply in soils. As three ginseng fields whose histories were known were analyzed, the PCR assay resulted as our expectation derived from the field information. The direct PCR method will be utilized as a reliable and rapid tool for detecting and monitoring C. destructans in ginseng fields.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.201-209
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• 2007

The CFDA (6-carboxyfluorescein diacetate) direct viable count method and plate count (PC) method using conventional nutrient broth (NB) medium and $10^{-2}$ diluted NB (DNB) medium were applied to samples collected from Mt. Yongdoo In Andong, in an effect to determine the number of living bacteria pine mushroom forest soil. The number of living bacteria determined via plate count in NB medium comprised $5{\sim}8%$ of the CFDA direct viable count, and the bacteria in the DNB medium comprised $40{\sim}47%$. This result indicated that viable but nonculturable (VBNC) bacteria existed in the pine mushroom forest soil at a high percentage. The phylogenetic characteristics of the VBNC bacterial populations in the samples of pine mushroom (Tricholoma matsutake) forest soil were analyzed via the direct extraction of DNA and 16S rDNA-ARDRA. The 115 clones from pine mushroom forest soil were clustered into 31 different RFLP phylotypes by ARDRA. Based on the 16S rDNA sequences, the 31 ARDRA clusters were classified into 6 phylogenetic groups: ${\alpha}-,\;{\beta}-,\;{\gamma}-Proteobacteria$, Acidobacteria, Actinobacteria and Firmicutes. Among these bacterial populations, approximately 85% were classified as members of phylum Acidobacteria. The Acidobacteria phylum was shown to exist abundantly in the pine mushroom forest soil.