scholarly journals Molecular techniques for the assessment of Cr (VI) reduction by Bacillus thuringiensis

2021 ◽  
Vol 26 (2) ◽  
Author(s):  
Deisy L. Guerrero-Ceballos ◽  
Eduardo Ibargüen-Mondragón ◽  
Pablo Fernández-Izquierdo ◽  
Jhonatan Pinta-Melo ◽  
Edith Mariela Burbano-Rosero
Keyword(s):  
Bacillus Thuringiensis ◽  
River Water ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bioinformatic Analysis ◽  
Molecular Techniques ◽  
Restriction Enzyme Digestion ◽  
Molecular Identity ◽  
Gradient Gel Electrophoresis ◽  
16Srrna Gene

Effluent pollution with Cr (VI) is a worldwide environmental problem. In the Pasto River (southeastern, Colombia), previous studies reported contamination with this metal at points near tanneries. To establish the role of Bacillus thuringiensis in Cr (VI) reduction in water from Pasto River, experiments were carried out with untreated Pasto River water (treatment 1), sterile Pasto River water inoculated with B. thuringiensis (treatment 2), and unsterilized Pasto River water inoculated with B. thuringiensis (treatment 3). All experiments were conducted in bioreactors with a controlled temperature of 20 °C and constant agitation for 156 h. Samples of 20 mL were taken every 12 h from each treatment to track Cr (VI) reduction levels and to confirm microorganism identity via molecular methods involving denaturing gradient gel electrophoresis (DGGE), restriction enzyme digestion profiles (RFLP), and bioinformatic analysis. Cr (VI) reduction was higher in treatment 3 (99:42 %) as opposed to treatment 2 (76:12 %) and treatment 1 (74:46 %). The molecular identity of B. thuringiensis was determined via sequencing of the 16SrRNA gene, and RFLP assessments in all three treatments revealed B. thuringiensis profiles. Since B. thuringiensis was present in all three treatments trough time, Cr (VI) reduction can be attributed to this bacterium.

scholarly journals Diversity of methanogens in ruminants in Queensland

2008 ◽  
Vol 48 (7) ◽  
pp. 722 ◽  
Author(s):  
D. Ouwerkerk ◽  
A. F. Turner ◽  
A. V. Klieve
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Rumen Fluid ◽  
Bos Indicus ◽  
Methane Emissions ◽  
Molecular Techniques ◽  
Feed Conversion ◽  
Methanogen Community ◽  
Gradient Gel Electrophoresis ◽  
Reduce Emissions ◽  
Cattle And Sheep

Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.

scholarly journals MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR-DGGE, A TRADITIONAL MEXICANBEVERAGE

2015 ◽  
Vol 3 (3) ◽  
pp. 1-15
Author(s):  
Marcial-Quino J. ◽  
Garcia-Ocón B. ◽  
Mendoza-Espinoza J.A. ◽  
Gómez-Manzo S. ◽  
Sierra-Palacios E
Keyword(s):  
Gel Electrophoresis ◽  
Fermentation Process ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Beverage Samples ◽  
D2 Domain ◽  
Gradient Gel Electrophoresis ◽  
26S Rrna

Currently it is well known that yeasts play an essential role in the production of different beverages. In this paper, were identified some of the yeasts involved in the fermentation process of the pulque, a Mexican traditional beverage. Samples were collected from different regions of Mexico and yeasts were detected directly from samples without cultivation. Identifying the yeasts was obtained using amplification the D1/D2 domain of the 26S rRNA gene and Denaturing Gradient Gel Electrophoresis (DGGE). The results of DGGE showed different profiles of bands in each of the analyzed samples, indicating the presence of several species of yeast, which was also confirmed by sequencing of the bands corresponding to the domain D1/D2, succeeded in identifying five species of yeasts. The results obtained in this work demonstrated that the technique used for identification of yeasts of pulque was efficient. Besides, the optimization of this method could also allow rapid identification of yeasts and help understand the role of these in the fermentation process of this beverage, as well as the isolation of strains of interest for biotechnological purposes such as production of ethanol or metabolites with nutraceutical activity.

Molecular Analysis of Spoilage-Related Bacteria in Pasteurized Milk during Refrigeration by PCR and Denaturing Gradient Gel Electrophoresis

2009 ◽  
Vol 72 (3) ◽  
pp. 572-577 ◽  
Author(s):  
HONGFEI HE ◽  
JIN DONG ◽  
CHIN NYEAN LEE ◽  
YONG LI
Keyword(s):  
Shelf Life ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Plate Count ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
Gradient Gel Electrophoresis ◽  
Aerobic Plate Count ◽  
Species Specific

Bacterial diversity in fluid milk products has been extensively studied in order to improve milk quality. Here, we illustrate the utility of viable counts and PCR–denaturing gradient gel electrophoresis (DGGE) for monitoring the microbial spoilage of pasteurized milk during shelf life. Five pasteurized milk samples stored at 4°C were examined at 10 and 5 days before expiration and on the expiration day. With bacterial DNA extracted directly from the samples, PCR-DGGE analysis indicated that Pseudomonas became dominant in four samples. Meanwhile, the aerobic plate count of these four samples exceeded the regulatory limit of 20,000 CFU/ml at 5 days before expiration, and the rapid psychrotrophic count markedly surpassed the aerobic plate count on the expiration day. Streptococcus and Buttiauxella spp. were detected in several samples. Sequence analysis of DGGE fragments revealed high diversity among Pseudomonas spp. in the milk samples. P. putida and P. migulae grew to high numbers during refrigerated storage. Further identification of Pseudomonas at the species level was facilitated by PCR and multiplex PCR using species-specific primers; consequently, P. fluorescens and P. fragi were observed. These results highlight an important role of Pseudomonas in the shelf life of pasteurized milk.

Quantification and genotyping of Cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis

2006 ◽  
Vol 54 (3) ◽  
pp. 119-126 ◽  
Author(s):  
Y. Masago ◽  
K. Oguma ◽  
H. Katayama ◽  
S. Ohgaki
Keyword(s):  
River Water ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Detection Method ◽  
Geometric Mean ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Cryptosporidium Spp ◽  
Gradient Gel Electrophoresis ◽  
Quenching Probe

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40 L of river water (geometric mean =35%, standard deviation =8.7%). Quenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/100 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.

Novel bacteria associated with Arctic seashore lichens have potential roles in nutrient scavenging

2014 ◽  
Vol 60 (5) ◽  
pp. 307-317 ◽  
Author(s):  
Margrét Auður Sigurbjörnsdóttir ◽  
Starri Heiðmarsson ◽  
Anna Rut Jónsdóttir ◽  
Oddur Vilhelmsson
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Symbiotic Association ◽  
Bacterial Strains ◽  
Rdna Sequencing ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Mutualistic Association ◽  
Modest Level

While generally described as a bipartite mutualistic association between fungi and algae or cyanobacteria, lichens also host diverse and heretofore little explored communities of nonphototrophic endolichenic bacteria. The composition and possible roles of these bacterial communities in the lichen symbiotic association constitute an emerging field of research. Saxicolous (rock-dwelling) seashore lichens present an unusual environment, characterized by rapid fluctuations in temperature, salinity, exposure to solar radiation, etc. The present study focuses on the bacterial biota associated with 4 species of crustose, halophilic, saxicolous seashore lichens found in northern Iceland. A denaturing gradient gel electrophoresis based characterization of the composition of the lichen-associated microbiotas indicated that they are markedly lichen-species-specific and clearly distinguishable from the environmental microbiota represented by control sampling. A collection of bacterial strains was investigated and partially identified by 16S rDNA sequencing. The strains were found to belong to 7 classes: Alphaproteobacteria, Bacilli, Actinobacteria, Flavobacteria, Cytophagia, Sphingobacteria, and Gammaproteobacteria. Several isolates display only a modest level of similarity to their nearest relatives found in GenBank, suggesting that they comprise previously undescribed taxa. Selected strains were tested for inorganic phosphate solubilization and biodegradation of several biopolymers, such as barley β-glucan, xylan, chitosan, and lignin. The results support a nutrient-scavenging role of the associate microbiota in the seashore lichen symbiotic association.

Mitochondrial Mutations as Molecular Markers for Minimal Reidual Disease (MRD) in Acute Myeloid Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2996-2996
Author(s):  
Alexander A. Morley ◽  
Scott A. Grist ◽  
Xiao J. Lu ◽  
Katrina Patsouris
Keyword(s):  
Acute Myeloid Leukemia ◽  
Molecular Markers ◽  
Myeloid Leukemia ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Residual Disease ◽  
Molecular Techniques ◽  
Leukemic Cells ◽  
Mitochondrial Mutations ◽  
Gradient Gel Electrophoresis ◽  
Acute Myeloid

Abstract Measurement of minimal residual disease (MRD) is being increasingly used in haematological malignancies in order to assess prognosis and decide on treatment. However for some patients, including many patients with acute myeloid leukemia (AML), molecular techniques for MRD measurement cannot be performed owing to lack of a molecular marker. We have detected mitochondrial mutations (MM) in the D loop of the mitochondrial genome of the leukemic cells in approximately 40% of patients with AML and have investigated 2 techniques - denaturing gradient gel electrophoresis (DGGE) and single nucleotide primer extension (SNUPE) - to detect and quantify them. Mixing experiments showed that DGGE had a sensitivity of approximately 0.5% for detection of MM, and it was able to detect leukemia in remission marrow in 5 of 6 patients with AML. When present in a patient, MM are usually multiple. We therefore tested a 2-step strategy, as shown in the figure, which involved initial enrichment by PCR using primers directed at 1 or 2 flanking mutations followed by DGGE to detect an inner mutation. In 2 mixing experiments this 2-step strategy increased sensitivity of detection down to 0.0001% (this level of detection was evident in the original gel photo). Figure Figure SNUPE was more sensitive than DGGE and in 3 mixing experiments it showed a sensitivity of 0.02–0.05%. Four patients with AML have now been studied by SNUPE and the levels of MRD in the marrow documenting morphological remission at the end of induction therapy were 2.8%,16.1%,40.8% and 15.7%. The relatively high levels of MRD as measured by PCR in 3 patients suggest that some of the leukaemic cells at the end of induction may be more mature than blasts and thus not identifiable by morphology. While questions remain to be answered, mitochondrial mutations are promising molecular markers for quantifying MRD in AML and possibly other haematological disorders

scholarly journals Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

2001 ◽  
Vol 67 (7) ◽  
pp. 2942-2951 ◽  
Author(s):  
Beatriz Dı́ez ◽  
Carlos Pedr�s-Ali� ◽  
Terence L. Marsh ◽  
Ramon Massana
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Phylogenetic Groups ◽  
Gradient Gel Electrophoresis ◽  
Phylogenetic Identification ◽  
Phylogenetic Composition ◽  
Primer Sets

ABSTRACT We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

scholarly journals Exploring the Role of Microorganisms in the Disease-Like Syndrome Affecting the Sponge Ianthella basta

2010 ◽  
Vol 76 (17) ◽  
pp. 5736-5744 ◽  
Author(s):  
Heidi M. Luter ◽  
Steve Whalan ◽  
Nicole S. Webster
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Disease Process ◽  
Community Analysis ◽  
Visual Assessment ◽  
Physical Contact ◽  
Brown Spot ◽  
Group I ◽  
Gradient Gel Electrophoresis ◽  
Affected Tissue

ABSTRACT A disease-like syndrome is currently affecting a large percentage of the Ianthella basta populations from the Great Barrier Reef and central Torres Strait. Symptoms of the syndrome include discolored, necrotic spots leading to tissue degradation, exposure of the skeletal fibers, and disruption of the choanocyte chambers. To ascertain the role of microbes in the disease process, a comprehensive comparison of bacteria, viruses, fungi, and other eukaryotes was performed in healthy and diseased sponges using multiple techniques. A low diversity of microbes was observed in both healthy and diseased sponge communities, with all sponges dominated by an Alphaproteobacteria, a Gammaproteobacteria, and a group I crenarchaeota. Bacterial cultivation, community analysis by denaturing gradient gel electrophoresis (Bacteria and Eukarya), sequencing of 16S rRNA clone libraries (Bacteria and Archaea), and direct visual assessment by electron microscopy failed to reveal any putative pathogens. In addition, infection assays could not establish the syndrome in healthy sponges even after direct physical contact with affected tissue. These results suggest that microbes are not responsible for the formation of brown spot lesions and necrosis in I. basta.

scholarly journals Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

2000 ◽  
Vol 66 (10) ◽  
pp. 4372-4377 ◽  
Author(s):  
Bo Normander ◽  
Jim I. Prosser
Keyword(s):  
Community Composition ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Populations ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

ABSTRACT An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

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