Interlaboratory Evaluation of Different Extraction and Real-Time PCR Methods for Detection of Coxiella burnetii DNA in Serum

In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever

UVC-induced lysis and detritus production of Oscillatoria limnetica in a two-stage continuous-flow system

In order to study in model systems the role of heterotrophic micro-organisms in the aquatic microbial food web, a natural food source consisting of senescent primary producer cells is indispensable. A two-stage continuous-flow system with the ability to produce detritus continuously is presented. In the first stage, a primary producer, i.e. the cyanobacterium Oscillatoria limnetica, was cultured. The second stage was fed with UVC-killed primary producer cells from the first stage. A control second stage received living primary producer cells. Both second stages were filled with prefiltered lake water in order to provide a natural inoculum of bacteria. During a 2 month experiment, the system was tested for its ability to produce detritus (first stage of detritus, i.e. lysis of the cell material). Chlorophyll a concentrations proved to be adequate for calculating specific lysis rates. Very high specific lysis rates were found after killing the cells with UVC as compared to the control stage. [KEYWORDS: Phytoplankton detritus; lake phytoplankton; oligotrophic lake; fresh-water; decomposition; bacteria; dynamics; algae]

Sequence analysis of the its-2 region: a tool to identify strains of Scenedesmus (Chlorophyceae)

The genetic distances between several strains of Senedesmus obliquus (Turp,) Kutz,, S, acutus Hortobagyi, and S, naegelii Chod. calculated from ITS-2 sequences were found to be smaller than the genetic distances within other strains of Scenedesmus-that is, in S, acuminatus (Lagerh,) Chod, and S, pectinatus Meyen. These results confirm that the studied strains were not properly identified and should be renamed S, obliquus, as already suggested in other studies. [KEYWORDS: ITS, phylogeny, Scenedesmus acutus, Scenedesmus naegelii, Scenedesmus obliquus]

Revealing genetic diversity of eukaryotic microorganisms in aquatic environments by denaturing gradient gel electrophoresis

A new Eucarya-specific 18S rDNA primer set was constructed and tested using denaturing gradient gel electrophoresis to analyze the genetic diversity of eukaryotic microorganisms in aquatic environments. All eukaryal lines of descent exhibited four or fewer nucleotide mismatches in the forward primer sequence, except for the microspora line of descent. The reverse primer annealed to a more conserved region with fewer than two nucleotide mismatches. Genomic DNA from test organisms with different numbers of nucleotide mismatches were amplified to test primer specificity. Relatively low annealing temperatures allowed the amplification of sequences with up to four nucleotide mismatches while still maintaining specificity for the eukaryal domain. Denaturing gradient gel electrophoresis was used to separate similarly sized PCR products of environmental samples, and the obtained banding patterns were converted to a binary format for statistical comparisons. Cluster analysis of these patterns showed similar results to a cluster analysis based on environmental variables. This approach provides an analytical tool to study the population structure and molecular ecology of eukaryotic microbial communities inhabiting aquatic environments. [KEYWORDS: 18S rRNA; aquatic environments; community structure; DGGE; Eucarya; genetic diversity; nucleotide mismatches; Southern blot hybridization 16s ribosomal-rna; polymerase chain-reaction; marine picoplankton; flow-cytometry; identification; fragments; bacteria; probes; phytoplankton; populations]

The impact of photoperiodicity on hatching of Loligo vulgaris and Loligo forbesi

The influence of photoperiodicity on hatching of Loligo forbesi and Loligo vulgaris embryos was investigated under different experimental light-dark (LD) conditions. The transition from light to dark stimulated hatching and functions as a "Zeitgeber" or synchronizer. Independent of the timing and duration of the dark period most embryos hatched soon after termination of the light period. Embyros which had developed in constant light, showed no hatching rhythm at all. If these embryos were exposed to a dark shock most embryos hatched soon after the onset of darkness. A twilight shock, in which the light was reduced by 50% (i.e. 50 µE s -1m-²), could not simulate hatching. Embryos which were kept from stage X on in an artifically controlled LD cycle, preferentially hatch in a period which coincides with the period at which darkness usually occured when placed in constant illumination from stage XX onwards

UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically effective dose BE(DNA300nm)) of 1.05 kJ m(-2) caused detectable damage in about 20% of the exposed population. In the period after the UV-B treatment, when the culture was exposed to PAR (450 mu mol m(-2)s(-1)) only, thymine dimers were removed; after 8 hours none of these photoproducts remained. Cellular DNA content measurements and quantification of the fraction of recently divided cells revealed that the DNA synthesis rate as well as the rate of cell division were reduced during and shortly after UV-B exposure. Apparently, UV-B irradiation extends the cell cycle of Cyclotella sp. in the S (DNA synthesis) phase until the dimers are removed. [KEYWORDS: cell cycle; DNA damage; flow-cytometry; marine diatom; thymine dimers; UV-B Ultraviolet-radiation; antarctica; dosimeter; dimers; ocean;cells]

Detritus-dependent development of the microbial community in an experimental system: qualitative analysis by denaturing gradient gel electrophoresis

Correlations between the biomass of phytoplankton and the biomass of bacteria and, between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the "microbial loop." To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used. to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities,the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members:of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined. [KEYWORDS: ALGAL EXTRACELLULAR PRODUCTS; DISSOLVED ORGANIC-CARBON; FRESH-WATER; BACTERIAL UTILIZATION; PHYTOPLANKTON; MARINE; BACTERIOPLANKTON; DECOMPOSITION; POPULATIONS; METABOLISM]