Capillary Electrophoresis Single-strand Conformation Research Articles

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate.

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

Reliable Procedure for Molecular Analysis of Airborne Microflora in Three Indoor Environments: An Office and Two Different Museum Contexts

AbstractBiological aerosols from air constitute a significant source of exposure to microorganisms in public places. Airborne microorganisms are involved in the development of certain respiratory symptoms, allergies, or infections among users and occupants. Various sampling instruments have commonly been used in aerobiology to collect bacteria and fungi suspended in the air. The objective of this study was to develop a reliable procedure for sampling in indoor public environments presenting different levels of occupancy, airborne bacteria and fungi to be subjected to molecular analysis (bacteria and fungi quantitative PCR, capillary electrophoresis single strand conformation polymorphism fingerprinting). Four different sampling devices were tested in situ in an office building (open‐plan type) and the sampling strategy chosen was tested in two museum contexts. In accordance with the drawbacks involved to our study (quantitative and qualitative aspects, cost, and overcrowding), cyclone device appeared to be most suitable. The results underline the effectiveness of this high‐volume aerosol sampling device for both qualitative and quantitative molecular analysis. Four in situ sampling collections were carried out in 1 day in the Louvre Museum to study quantitative and qualitative variations of airborne bacterial and fungal diversity. The quantitative results revealed a similar order of magnitude for the numbers of both bacteria and fungi. In the Louvre Museum, the samples yielded between 3.7 × 104 and 4.1 × 104 genome equivalent (GE) bacteria/m3 air and between 5.0 × 104 and 5.9 × 104 GE fungi/m3 air and in the Decorative Arts Museum between, 2.1 × 104 and 2.5 × 104 GE bacteria/m3 air and between 1.4 × 104 and 1.7 × 104 GE fungi/m3 air. The results also indicate that the dominant bacterial community displayed a stable structure over a short period of time whereas dominant eukaryotic airborne community appeared more variable.

Rapid adaptation of the bacterial community in the growing rabbit caecum after a change in dietary fibre supply

This work aimed to study the response of the growing rabbit caecal ecosystem (bacterial community and caecal environmental parameters) after a switch from a control to a low-fibre diet (LFD). A group of 160 rabbits were fed ad libitum a control diet (ADF: 20.4%) from weaning (36 days). At 49 days of age (day 0), 75 rabbits were switched to a LFD group (ADF: 10.7%), whereas 85 others (control group) remained on the control diet, for 39 days. Caecal contents were regularly sampled throughout the trial (60 rabbits per group). The bacterial community structure was characterized using CE-SSCP (capillary electrophoresis single strand conformation polymorphism) and total bacteria were quantified using real-time PCR. Redox potential (Eh), pH, NH3-N, volatile fatty acid (VFA) were measured in the caecum to characterize environmental parameters. The reduction of fibre in the diet modified the CE-SSCP profiles (P < 0.001) but not the diversity index (5.6 ± 0.8, ns). The number of 16S rRNA gene copies of total bacteria decreased (P < 0.01) in LFD rabbits compared with controls. In LFD rabbits, the caecal environment was less acid (+0.2 units; P < 0.01), more reductive (−11 mV; P < 0.05) and drier (+3.4 g 100 per g; P < 0.001), with an increase in NH3-N (+77%; P < 0.001) and a decrease in total VFA concentration (−17%; P < 0.001). We found significant correlations between the bacterial community, the quantity of bacteria and the caecal traits of the caecal ecosystem. Indeed, in both groups, the caecal traits barely constrained the total inertia of the CE-SSCP profile set (less than 14%), whereas total bacteria were positively related to total VFA, acetic acid and butyric acid levels, and Eh, and negatively related to pH. All the microbial and environmental modifications had occurred by day 2 and remained stable thereafter. These results suggest that the bacterial community in the growing rabbit caecum is able to adapt quickly after a change to in the dietary fibre supply to reach a new steady-state equilibrium.

Development and application of a functional CE-SSCP fingerprinting method based on [Fe–Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures

A Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) method based on functional [Fe–Fe]-hydrogenase genes was developed for monitoring the hydrogen (H 2)-producing clostridial population in mixed-culture bioprocesses. New non-degenerated primers were designed and then validated on their specific PCR detection of a broad range of clostridial hydA genes. The hydA-based CE-SSCP method gave a specific and discriminating profile for each of the Clostridium strains tested. This method was validated using H 2-producing mixed cultures incubated at temperatures ranging from 25 °C to 45 °C. The hydA CE-SSCP profiles clearly differed between temperatures tested. Hence, they varied according to variations of the H 2 production performances. The HydA sequences amplified with the new primer set indicated that diverse Clostridium strains impacted the H 2 production yields. The highest performances were related to the dominance of Clostridium sporogenes-like hydA sequences. This CE-SSCP tool offers highly reliable and throughput analysis of the functional diversity and structure of the hydA genes for better understanding of the H 2-producing clostridial population dynamics in H 2 dark fermentation bioreactors.

The role of sarcomere gene mutations in patients with idiopathic dilated cardiomyopathy

We investigated a Danish cohort of 31 unrelated patients with idiopathic dilated cardiomyopathy (IDC), to assess the role that mutations in sarcomere protein genes play in IDC. Patients were genetically screened by capillary electrophoresis single strand conformation polymorphism and subsequently by bidirectional DNA sequencing of conformers in the coding regions of MYH7, MYBPC3, TPM1, ACTC, MYL2, MYL3, TNNT2, CSRP3 and TNNI3. Eight probands carried disease-associated genetic variants (26%). In MYH7, three novel mutations were found; in MYBPC3, one novel variant and two known mutations were found; and in TNNT2, a known mutation was found. One proband was double heterozygous. We find evidence of phenotypic plasticity: three mutations described earlier as HCM causing were found in four cases of IDC, with no history of a hypertrophic phase. Furthermore, one pedigree presented with several cases of classic DCM as well as one case with left ventricular non-compaction. Disease-causing sarcomere gene mutations were found in about one-quarter of IDC patients, and seem to play an important role in the causation of the disease. The genetics is as complex as seen in HCM. Thus, our data suggest that a genetic work-up should include screening of the most prominent sarcomere genes even in the absence of a family history of the disease.

Role of environmental factors for the vertical distribution (0–1000 m) of marine bacterial communities in the NW Mediterranean Sea

Abstract. Bacterioplankton plays a central role in energy and matter fluxes in the sea, yet the factors that constrain its variation in marine systems are still poorly understood. Here we use the explanatory power of direct multivariate gradient analysis to evaluate the driving forces exerted by environmental parameters on bacterial community distribution in the water column. We gathered and analysed data from a one month sampling period from the surface to 1000 m depth at the JGOFS-DYFAMED station (NW Mediterranean Sea). This station is characterized by very poor horizontal advection currents which makes it an ideal model to test hypotheses on the causes of vertical stratification of bacterial communities. Capillary electrophoresis single strand conformation polymorphism (CE-SSCP) fingerprinting profiles analyzed using multivariate statistical methods demonstrated a vertical zonation of bacterial assemblages in three layers, above, in or just below the chlorophyll maximum and deeper, that remained stable during the entire sampling period. Through the use of direct gradient multivariate ordination analyses we demonstrate that a complex array of biogeochemical parameters is the driving force behind bacterial community structure shifts in the water column. Physico-chemical parameters such as phosphate, nitrate, salinity and to a lesser extent temperature, oxygen, dissolved organic carbon and photosynthetically active radiation acted in synergy to explain bacterial assemblages changes with depth. Analysis of lipid biomarkers of organic matter sources and fates suggested that bacterial community structure in the surface layers was in part explained by lipids of chloroplast origin. Further detailed analysis of pigment-based phytoplankton diversity gave evidence of a compartmentalized influence of several phytoplankton groups on bacterial community structure in the first 150 m depth.

Safum: statistical analysis of SSCP fingerprints using PCA projections, dendrograms and diversity estimators

AbstractThe program safum provides a smart interface to import, visualize and compare fingerprinting profiles, especially on capillary electrophoresis single strand conformation polymorphism data, in conjunction with basic statistical analysis tools. It includes principal component analysis, two‐ or three‐dimensional representations, dendrograms based on Euclidean distance, and easily exportable files for subsequent applications. safum is useful for the analysis of spatial or temporal sequences of microbial community fingerprints obtained with an ABI prism sequencer.

Single-strand conformation polymorphism analysis using capillary array electrophoresis for large-scale mutation detection

This protocol describes capillary array electrophoresis single-strand conformation polymorphism (CAE-SSCP), a screening method for detection of unknown and previously identified mutations. The method detects 98% of mutations in a sample material and can be applied to any organism where the goal is to determine genetic variation. This protocol describes how to screen for mutations in 192 singleplex or up to 768 multiplex samples over 3 days. The protocol is based on the principle of sequence-specific mobility of single-stranded DNA in a native polymer, and covers all stages in the procedure, from initial DNA purification to final CAE-SSCP data analysis, as follows: DNA is purified, followed by PCR amplification using fluorescent primers. After PCR amplification, double-stranded DNA is heat-denatured to separate the strands and subsequently cooled on ice to avoid reannealing. Finally, samples are analyzed by capillary electrophoresis and appropriate analysis software.

High‐efficiency multiplex capillary electrophoresis single strand conformation polymorphism (multi‐CE‐SSCP) mutation screening of SCN5A: a rapid genetic approach to cardiac arrhythmia

Mutations in the SCN5A gene coding for the alpha-subunit of the cardiac Na(+) ion channel cause long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, sick sinus node syndrome, progressive conduction disease, dilated cardiomyopathy and atrial standstill. These diseases exhibit variable expressivity, and identification of gene carriers is clinically important, particularly in sudden infant and adult death syndromes. The SCN5A gene comprises 28 exons distributed over 100 kbp of genomic sequence at chromosome 3p21. Disease-causing mutations are private and scattered over the DNA sequence, making it difficult to screen for specific mutations. We developed a multiplex capillary-electrophoresis single-strand conformation polymorphism (Multi-CE-SSCP) mutation screening protocol on the ABI 3100 platform and applied it to 10 previously slab-gel SSCP identified mutations and SNPs and used it to identify one novel deletion. The method is highly efficient, with a turnover of 23 patients per 24 h and a false positive rate of 0.5% of the analyzed amplicons. Each variant has a particular elution pattern, and all 20 carriers of the H558R polymorphism out of 57 persons were correctly identified. We suggest that the method could become part of routine work-up of patients with suspicious syncope and of members of families with sudden unexplained death.

Analysis of TP53 gene mutations in human lung cancer: Comparison of capillary electrophoresis single strand conformation polymorphism assay with denaturing gradient gel electrophoresis and direct sequencing

Introduction: Automated capillary electrophoresis single strand conformation polymorphism (CE-SSCP), denaturant gradient gel electrophoresis (DGGE), and direct sequencing were compared to investigate the benefits and sensitivity of each of the methods for detection of unknown TP53 mutations in human lung cancer. Methods: Twenty previously analyzed DNA samples from lung tumors were examined under dummy laboratory codes for occurrence of mutations of the TP53 gene. Results: Mutations were found in 17 samples; 15 (88%) of them were detected by DGGE, 16 (94%) by CE-SSCP and 12 (71%) by direct sequencing. One of the two mutations that remained undetected in DGGE was in fact outside the sequence area covered by DGGE screening, thus rendering DGGE and CE-SSCP equally efficient in mutation detection. Direct sequencing performed less well in finding mutations than the two other assays, as also shown previously. Conclusion: The study showed that CE-SSCP is a fast and highly reproducible method, which is considerably less laborious compared to DGGE, for screening of unknown TP53 mutations.

Spatial and temporal scales of variation in bacterioplankton community structure in the NW Mediterranean Sea

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 40:229-240 (2005) - doi:10.3354/ame040229 Spatial and temporal scales of variation in bacterioplankton community structure in the NW Mediterranean Sea Jean-François Ghiglione*, Marièle Larcher, Philippe Lebaron Observatoire Océanographique de Banyuls, Laboratoire Arago, Université Pierre et Marie Curie (Paris 6), Institut National des Sciences de l’Univers (INSU) CNRS-UMR-7621, BP 44, 66651 Banyuls-sur-mer Cedex, France *Email: ghiglione@obs-banyuls.fr ABSTRACT: An important query in marine microbial ecology is at what temporal and spatial scales variations in the structure of natural bacterial communities occur. Samples of different volumes were collected at different spatial (vertical and horizontal) and temporal (from hours to seasons) scales along a transect between a coastal station (26 m depth) and an offshore Microbial Observatory (1000 m depth) in the NW Mediterranean Sea. The structure of the bacterial communities was determined by capillary electrophoresis–single strand conformation polymorphism (CE–SSCP) fingerprinting of polymerase chain reaction (PCR)-amplified 16S rDNA. This technique is a powerful tool to compare natural microbial assemblages at different spatial or temporal scales. Similar bacterial assemblages were found up to 3.7 km from the coastal station, whereas significant changes were found over greater distances (from 9.3 to 33.3 km). Although the bacterial community structure did not change with depth at coastal and shallow stations, vertical changes were found at deeper stations, most likely due to vertical variations in physico-chemical and biogeochemical parameters. Temporal changes were mainly related to environmental variations that occurred at a seasonal scale and during phytoplankton blooms. Finally, we suggest that long-term studies, at least in the NW Mediterranean Sea, should involve a minimum sampling time scale of 2 wk and a shorter time-scale when environmental changes are detected by the real-time monitoring of a few basic parameters (i.e. fluorescence, temperature, salinity). Sampling strategies should also include different depths depending on the vertical structure of the water column, based on the same basic parameters. KEY WORDS: Bacteria · CE–SSCP · Community structure · Sampling strategy · Microbial observatory Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 40, No. 3. Online publication date: October 18, 2005 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2005 Inter-Research.

Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis.

A specific reverse transcription-polymerase chain reaction (RT-PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.

Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector

Mutations of K-ras gene play an important role in neoplastic progression. The capillary electrophoresis–single strand conformation polymorphism (CE–SSCP) technique is available for the detection of gene mutations. Using an automated capillary electrophoresis with short-chain linear polyacrylamide, after denaturation of PCR products, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under a constant voltage of 8 kV. Of 16 specimens of lung cancer tissue, two specimens were found to have abnormal peaks in the electrophoretogram. CE–SSCP is rapid, automated, and has high performance.