PCR amplification of crude microbial DNA extracted from soil

Yeates, Christine; Gillings, Michael R.; Davison, Annette; Altavilla, Nanda; Veal, Duncan

doi:10.1046/j.1472-765x.1997.00232.x

Cited by 82 publications

(68 citation statements)

References 4 publications

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“…The amount of surplus H 2 O 2 from the 25 ml of the filtrate was determined by titration with 0.02 mol l -1 KMnO 4 . DNA extraction and PCR-DG-DGGE At 0, 15, 30, and 45 days, the total genomic DNA of the soil was extracted from each soil sample (0.5 g dry equivalent weight), as previously described (Yeates et al 1997). A 16S rRNA gene fragment was amplified by PCR, using the bacterium-specific primers 341 F-GC and 534 R (Cremonesi et al 1997).…”

Section: Enzymatic Activities Assaymentioning

confidence: 99%

Effect of decabromodiphenyl ether (BDE 209) on soil microbial activity and bacterial community composition

Zhu

Liu

Zou

et al. 2010

World J Microbiol Biotechnol

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In many areas of the world, polybrominated diphenyl ethers are ubiquitous due to their use as fire retardants. It is known that the hydrophobic characteristics of PBDEs cause them to sink in soil and sediment, yet their effect on microbes within the soil is not well understood. In this study, soil was treated with 1, 10, and 100 mg kg -1 BDE 209 for up to 45 days. Treatment effects on soil enzymatic activities for urease and catalase were evaluated. The impact on the microbe community structure was estimated using denaturing gradient gel electrophoresis after polymerase chain reaction amplification of total genomic DNA, using bacterial variable V3 region targeted primers. The effects on the soil microbial community size and major bacterial groups were evaluated using fluorescence in situ hybridization analysis. Forty-five days after the addition of BDE 209, urease activity was suppressed by BDE 209, even at a concentration of 1 mg kg -1 . Catalase activity increased in the samples containing lower concentrations of BDE 209, but was suppressed in samples containing higher concentrations. The bacterial community also varied in response to the addition of BDE 209, and the variation of community composition differed among treatments. In addition, a, b and c subclass proteobacteria decreased in the group of 100 mg kg -1 BDE 209 spiked soil after 45 days of treatment. Throughout the experiment, no BDE 209 degradation was detected under darkness. These observations demonstrated that BDE 209 in soil, although of low bioavailability, had an adverse impact on the structure and function of the soil microbial community and microbial processes.

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Section: Enzymatic Activities Assaymentioning

confidence: 99%

Effect of decabromodiphenyl ether (BDE 209) on soil microbial activity and bacterial community composition

Zhu

Liu

Zou

et al. 2010

World J Microbiol Biotechnol

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“…Although these methods have proven to be very useful for determining whether or not waters are contaminated with parasites, they cannot distinguish among the different species or genotypes. To this end, PCR assays have been developed (33), yet the efficiency of amplification techniques is often reduced by the presence of inhibitory substances in water samples, such as humic and fulvic acids (37,42). In Italy, there are few published data on the prevalence of Giardia and Cryptosporidium in wastewaters.…”

mentioning

confidence: 99%

Giardia Cysts in Wastewater Treatment Plants in Italy

Cacciò¹,

Giacomo²,

Aulicino

et al. 2003

Appl Environ Microbiol

160

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Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O 2 and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P ‫؍‬ 0.05, analysis of variance). To characterize the cysts at the molecular level, the ␤-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater.

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“…DNA was extracted using the method suggested by Yeates et al (1997) and purified subsequently with CTAB (Manual Method 1, MM1) and PEG 4000 (Manual Method 2, MM2) . …”

Section: Direct Lysis and Purification Methodsmentioning

confidence: 99%

Direct Lysis Glass Milk Method of Genomic Dna Extraction Reveals Greater Archaeal Diversity in Anaerobic Biodigester Slurry as Assessed Through Denaturing Gradient Gel Electrophoresis

Verma¹,

Vasudevan²,

Kashyap³

et al. 2018

JEBAS

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DNA extraction from anaerobic biodigester slurry is a critical step in all phylogenetic and metagenomic approaches to characterize highly diverse biodigester ecosystem, but little is known about the efficiency of different extraction procedures and their impact on subsequent analyses of microbial communities. The assessment of performance differences, therefore, is a concrete step towards the determination of optimal DNA extraction method for biodigester slurry, which can provide a more reliable comparison of the metaanalysis results obtained in different conditions. Here, we report a highly efficient direct lysis genomic DNA extraction method (Glass milk method) from the slurry of an anaerobic biodigester. This method was compared with five commercially available DNA extraction kits and two different manual methods with regard to DNA extraction, purification efficiencies and representation of archaeal diversity through PCRDenaturing Gradient Gel Electrophoresis (DGGE). Results revealed that commercial kits yielded significantly lower DNA concentrations than manual methods. Moreover, interpretations of bacterial community structure based on analysis of PCR-DGGE banding pattern of 16S rRNA gene, ShannonWiener and Simpson's indices of diversity and multidimensional scaling suggested that manual methods of DNA isolation revealed far greater archaeal diversity as compared to the commercial kits. Further, in realtime PCR analysis, a methanogens-specific 16S rRNA gene concentration was observed more in manual methods than in commercial kits. The glass milk DNA extraction method proposed here is very useful in pursuing phylogenetic and metagenomic approaches to characterize the highly complex anaerobic biodigester ecosystem with greater reliability and precision.

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PCR amplification of crude microbial DNA extracted from soil

Cited by 82 publications

References 4 publications

Effect of decabromodiphenyl ether (BDE 209) on soil microbial activity and bacterial community composition

Effect of decabromodiphenyl ether (BDE 209) on soil microbial activity and bacterial community composition

Giardia Cysts in Wastewater Treatment Plants in Italy

Direct Lysis Glass Milk Method of Genomic Dna Extraction Reveals Greater Archaeal Diversity in Anaerobic Biodigester Slurry as Assessed Through Denaturing Gradient Gel Electrophoresis

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