Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

<p>Abstract</p> <p>Background</p> <p>The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean^®Fecal DNA Isolation Kit, M; QIAamp^®DNA Stool Mini Kit, Q; FastDNA^®SPIN Kit, FSp; FastDNA^®SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.</p> <p>Method</p> <p>Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.</p> <p>Results</p> <p>Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.</p> <p>Conclusion</p> <p>We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.</p

Soil enzyme activity patterns in organic and conventional cultivation

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The impact of crop plant cultivation and peat amendment on soil microbial activity and structure

v2004o

Enterococcus rivorum sp. nov., from water of pristine brooks

A significant number of Enterococcus strains from pristine waters of two brooks in Finland formed a distinct cluster on the basis of whole-cell protein fingerprinting by one-dimensional SDS-PAGE. The strains shared the following characteristics. Cells were ovoid, Gram-positive-staining and non-spore-forming, appearing singly or in pairs or chains. They were facultatively anaerobic and catalase-negative. Growth in broth containing 6.5% NaCl or at 45 degrees C was weak or absent. Production of D antigen was variable. The strains tolerated 60 degrees C for 30 min, 40% bile and tellurite, hydrolysed aesculin strongly and gelatin weakly, produced no acid from hippurate and did not reduce it, grew weakly at 10 degrees C, showed a strong reaction for the Voges-Proskauer test and produced acid from methyl alpha-D-glucoside, mannitol, sorbitol and sucrose, with weak or no production of acid from methyl alpha-D-mannoside, L-arabinose, gluconate and L-xylose. Several of the strains were selected for identification on the basis of sequencing of almost the whole 16S rRNA gene and partial atpA and pheS genes and of (GTG)(5)-PCR fingerprints. Partial atpA and pheS gene sequencing was also performed for those type strains of Enterococcus species without available sequences in the database. The pristine brook isolates formed a novel species, for which the name Enterococcus rivorum sp. nov. (type strain S299(T) =HAMBI 3055(T) =LMG 25899(T) =CCM 7986(T)) is proposed. On the basis of 16S rRNA gene sequence similarity, E. rivorum sp. nov. is related to the Enterococcus faecalis genogoup. It is distinguished from described Enterococcus species on the basis of 16S rRNA, atpA and pheS gene sequences and whole-cell protein and (GTG)(5)-PCR fingerprints. It is most closely related to E. faecalis, but DNA-DNA hybridization confirms it to represent a novel species

Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation

Abstract: In the conventional Cultivation of potatoes, weed control and the control of potato late blight caused by Phytophthora infestans are carried out by the application of herbicides and fungicides. We investigated the impacts of the herbicides metribuzin and linuron and the fungicide fluazinam oil soil microbiota in microcosms, in mesocosms and in the field. The toxicity of each pesticide in Solution was assessed using the luminescent bacteria test and in soil by a solid phase modification. In microcosm tests, the microbial activity and biomass were estimated by measuring several soil enzyme activities together with soil ATP content. In the mesocosm tests, the separate addition of each pesticide and the Simultaneous use of all the pesticides were investigated. We monitored the impacts on ten different soil enzyme activities and Measured soil toxicity with the luminescent bacteria test separately in the 5 cm top layer and in the layer from 5 to about 20 cm below the surface. During one season, the impact of the use of pesticides was monitored in the field in plots receiving pesticides for the third consecutive year and in control plots Cultivated without the use of pesticides for the 3 preceding years. The pesticide concentrations were monitored in each experiment. The luminescent bacteria toxicity test revealed a strong inhibition by fluazinam. In microcosms the herbicides increased several enzyme activities but metribuzin inhibited luminescence in the bacterial test. Fluazinam was highly toxic in microcosms. In the mesocosm with combined use of pesticides, decreased activities of some enzymes were observed first in the surface soil and at harvesting also deeper in the soil. In the mesocosm experiment with separate use of pesticides, less impacts were observed. In the field experiment the pesticides decreased seven enzyme activities, when calculated per soil fresh weight but activities of four enzyme decreases if calculated per soil organic matter. Controlling weeds by herbicides decreased weed growth and the decreases in enzyme activities were interpreted to be due to the lack of the stimulating impact of weed roots. A strong inhibition in the soil toxicity test and continuing bioavailability of fluazinam were detected throughout the experiments even after winter in the field. (c) 2008 Elsevier B.V. All rights reserved.v2009oktat ta