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To evaluate different types of sample storage and DNA extraction techniques for the real-time PCR quantification of cyanobacteria in water.
Two different filter types for the cell harvest of Microcystis sp. and Planktothrix spp. that were either freeze-dried or stored frozen, and two different methods for DNA extraction were compared. DNA extraction was achieved by standard phenol-chloroform extraction or by a faster commercially available purification kit (DNeasy ®, Quiagen). In general there was good agreement between the cell number equivalents of phycocyanin (PC) genotypes that were estimated using the Taq nuclease assay (TNA) between both filter types and the storing of samples. The standard DNA extraction procedure gave higher numbers of PC genotypes when compared to the DNeasy procedure. TNA results obtained from Planktothrix from natural samples extracted with the standard procedure revealed a significant correlation with the cell numbers estimated via the microscope.
Freeze-drying of samples gives quantifiable data. The standard DNA extraction is considered to be the most reliable and accurate, although the DNeasy procedure is useful for early warning monitoring.
Application of quantitative genotype analysis in cyanobacteria from freeze-dried samples collected during recent and past sampling programs.
Cyanobacteria of the genera Microcystis sp. and Planktothrix spp. are frequently found in freshwater. Cyanobacteria are considered as important source of organic toxic compounds deteriorating drinking water quality worldwide (WHO 2004). Typically, non-toxic strains and strains containing different toxins co-occur. Parallel to the elucidation of the genes that are involved in toxin synthesis (see Dittmann and Börner 2005 for a recent review) techniques have been developed in order to detect and quantify toxic genotypes directly in water. The real-time PCR technique, i.e. the Taq Nuclease Assay (TNA) has been introduced as tool for the quantification of toxic genotypes in water (Kurmayer and Kutzenberger 2003). The intensity of a genotype specific signal induced by a fluorescent TaqMan probe (i.e. the microcystin synthetase gene (mcy) of Microcystis sp.) is related to the signal specific of the total population (i.e. the phycocyanin operon of Microcystis sp.). Due to the binding of the primers and the probe, the TNA has been found to be highly specific for the target DNA and to produce reliable results even in the presence of complex background such as in field samples (Kurmayer and Kutzenberger 2003).
In the course of the EU-project PEPCY (Toxic and other bioactive peptides in cyanobacteria, QLRT-2001-02634, www.pepcy.de, 23 April 2005) a number of water bodies located within European countries were selected for quantifying toxin producing genotypes. Two problems arising from the sampling schedule were investigated: (i) how the field samples should be stored and posted and (ii) how DNA from cyanobacteria in field samples can be quantitatively extracted. A standard phenol-chloroform extraction procedure has been derived from established protocols (Franche and Damerval 1988) in an earlier study by Kurmayer et al. (2003) and has been shown to produce quantitative results (Kurmayer and Kutzenberger 2003). However, it is also considered to be time intensive regarding a large number of field samples and particularly for early warning monitoring a more time efficient and easy-to-use technique for DNA extraction would be required. Such a new technique needs to be applicable to all morphologies of cyanobacteria, for example Microcystis is growing as single cells embedded in mucilage while Planktothrix is forming rigid filaments consisting of cells tightly attached to each other.
In this study we tested the TNA results obtained for (i) using two types of filters for cell harvest which were either freeze-dried or frozen and (ii) different amounts of cells from strains of Microcystis sp. and Planktothrix spp. extracted using a commercially available DNA extraction kit (DNeasy, Quiagen) as opposed to the standard phenol-chloroform extraction procedure. TNA was specifically designated for the intergenic spacer region of the phycocyanin operon (PC-IGS).
For establishing the calibration curve P. rubescens strain PCC7821 (L. Gjersjøen, NO) was grown in BG11 medium (Rippka 1988) at 20°C and 30μE (continuous light, Philips TLD, 36W/965) and harvested under logarithmic growth phase conditions using low vacuum filtration onto glass fiber filters (BMC, Ederol, Vienna). As for all experiments aliquots were fixed with formaldehyde (2% final concentration) for cell enumeration in parallel to cell harvesting. Because all cyanobacteria contain the PC-IGS, DNA from Microcystis sp. strains HUB53 and HUB524 (Pehlitzsee, D) and Synechococcus strains MW15#2SUB (Mondsee, AT) was extracted and used as background DNA in order to test the specificity of the new TNA designed for PC-IGS of P. agardhii and P. rubescens.
Planktothrix strain PCC7821 and Microcystis strain HUB524 were grown and harvested as described above for comparing the TNA results between DNA extracts obtained (i) from glass fiber filters (Ederol, Vienna, type BMC, ø 47 mm, which is equivalent to the GF/C filter type, Whatman, Kent, Great Britain) vs. membrane filters (ME, Schleicher and Schüll, RC55, ø 50 mm) and (ii) from freeze-dried samples vs. frozen samples. Filters were freeze-dried using a speed vac (vacuum centrifuge concentrator 5301, Eppendorf, Hamburg, Germany) or were directly stored wet frozen at −20°C. There were three parallels per treatment. To compare DNA extraction methods, the following strains were used: P. rubescens: No6 (Mondsee, AT), No108 (Irrsee, AT), No61 (Schwarzensee, AT), No67 (Wörthersee, AT), No34 (Ammersee, D), No75 (Zürichsee, CH); P. agardhii: No31/1 (Wannsee, D), PCC7805 (Veluwermeer, NL), CCAP1459/17 (Blelham Tarn, UK), CYA126/8 (L. Langsjön, FI); M. aeruginosa: M. aeruginosa Hofbauer and M. flos-aquae (Neusiedlersee, AT), HUB524, HUB53, P461 (Pehlitzsee), W368, W334, W75, W61 (Wannsee, D), PCC7806 (Braakman Reservoir, NL). These strains were grown as described above and harvested during the stationary phase.
Formaldehyde-fixed samples were stained with DAPI according to standard protocols and filtered onto 0.2-μm-pore-size polycarbonate filters (GTBP, ø 25 mm, Millipore, Ireland). At least 400 cells per transect were counted at 1,000× (Axioplan, Zeiss, Germany) and cells per ml were calculated by way of extrapolation. Field samples integrated from the surface down to 8 m of depth from Lake Wannsee (Berlin, Germany) from June 1999 to October 2000 were analyzed for cell numbers using the inverted microscope as described (Kurmayer et al. 2003).
Filters were freeze-dried and stored at −20°C until DNA extraction. The standard phenol-chloroform DNA extraction procedure was performed as described in Kurmayer et al. (2003) and used for all analyses. For comparing extraction methods isolation of DNA was also achieved using the DNeasy Plant system (Quiagen, VWR, Vienna). Cells were not incubated in liquid nitrogen before digestion by incubating the filter using a provided lysis buffer (containing Rnase A) for 10 min at 65°C. Subsequently polysaccharides and proteins were precipitated and incubated 5 min on ice. The undigested cellular debris was removed using the QIAshredder spin column. A binding buffer (containing ethanol) was added to the cleared lysate and the DNA was purified using a DNeasy mini spin column (Quiagen 2003). In order to exclude the possibility of saturation of the extraction columns by excess amounts of DNA strain HUB524 (15×106 cells ml−1) was harvested from stationary phase on GF/C filters (5.4×107 cells filter−1) and extracted in 1, ½, ¼, 1/10, 1/100, 1/200, 1/400, 1/800 amounts. If saturation of the extraction column occurred it was expected that yields on a per cell basis should improve when cell numbers per extraction column were decreased.
Primers and the TaqMan probe that were specifically bound to PC-IGS of Microcystis sp. were designed by Kurmayer and Kutzenberger (2003). Primers and probes that were specifically bound to PC-IGS of Planktothrix rubescens and P. agardhii (sensu Suda et al. 2002) were designed during this study. The following PC-IGS sequences from cyanobacteria were aligned using Clustal W 1.8: Synechococcus sp. (AY151231, AY151230, AY151224, AY151222, AF223463, AF223465, AF223464), Synechocystis sp. (AJ003180), Chroococcus sp. (AJ003188), Microcystis sp. (strain PCC7806, AF195177), Planktothrix spp. (AJ558135, AJ132279, AJ131820, AJ558138, AJ558140, AJ558160, AJ401186, AJ401185), Aphanizomenon sp. (AJ243968), Lyngbia sp. (AJ401187), Nostoc sp. (NC 003272). In addition PCR for the PC-IGS region of the Microcystis strains HUB524, HUB53 was performed using the primers designed by Neilan et al. (1995) and the PCR products were sequenced and submitted to the DDBJ/EMBL/GenBank (AJ965489, AJ965490). Planktothrix specific primers were designed from gene regions that are homogeneous within P. rubescens and P. agardhii but are variable enough when compared to other genera: PlPC fwd: 5′-GAGCAGCACTGAAATCCAAG-3′, PlPC rev: 5′-GCTTTGGCTGCTTCTAAACC-3′). The TaqMan probe (PlPC 5′-6FAM-TTTGGCTTGACGGAAACGACCAA XT--PH-3′) had a fluorescent reporter dye (6-carboxyfluorescein) covalently attached to the 5′ end (5′- FAM) and a 3′- TAMRA fluorescent quencher dye (6-carboxytetramethylrodamine). The size of the amplification product was 72 bp. TNAs were performed using a GeneAmp 5700 sequence detection system (ABI, Vienna, Austria) as described (Kurmayer and Kutzenberger 2003). The primer and probe concentrations were optimized following the manufacturer′s instructions. The 25 μl reaction mix for PC-IGS of Planktothrix consisted of 12.5 μl (2×) TaqMan Universal PCR Master Mix (ABI, Vienna), 50 nM forward primer, 300 nM reverse primer, 100 nM TaqMan probe, 5 μl of template and 6.5 μl Millipore water. Each measurement was done in triplicate. Standard curves were established by relating the cell numbers to the measured threshold cycle (the cycle number where fluorescence exceeds the threshold set at 0.1). PC-IGS of Planktothrix PCC7821 was analyzed by comparing the calibration curve in the absence and presence of background DNA (Microcystis, Synechococcus) which was added in two concentrations, 1.7 × 104 and 1.7 × 105 cells per template consisting of 13% HUB53, 11% HUB524 and 76% MW15#2SUB.
The regression equation for the detection of PC-IGS using the TNA showed a significant linear curve between the amount of DNA in the template (expressed as cell number equivalents) and the Ct-value obtained: y = 38.20 –3.61× (R2 = 0.99, n = 6, P < 0.0001), where y is the Ct-value and x is the amount of DNA per template calculated in log10 cell number equivalents (data not shown). The dilution with the lowest detectable signal corresponded to one cell per template. The variation in Ct-values in the presence of both dilutions of the DNA background was small (ΔCt < 1) within the central region of the standard curve (40-40,000 cells template−1), but more pronounced towards higher (400,000 cells, ΔCt = 1.2) or lower cell numbers (< 1 cell, ΔCt = 3.4). A DNA concentration equivalent to 400 cells per template was found with the lowest deviation, and in further analysis all of the DNA extracts were diluted to this concentration.
Independent of the storage method and the filter type, the total variability in Ct-values obtained from standard DNA extracts was low and corresponded to the cell number determination in the microscope. At the day of cell harvest Plankthothrix PCC7821 had 5.3×104 cells ml−1 and Microcystis HUB524 had 1.7×106 cells ml−1. For strain PCC7821 Ct-values for ME filters wet frozen and ME freeze-dried were 23.5 – 24.6 (min-max) and 23.8 –25.4, each and for GF/C filters wet frozen and GF/C freeze-dried 25.4 –29.5 and 22.6 –26.5, each. For strain HUB524 Ct-values for ME filters wet frozen and ME freeze-dried were 29.2 –31.0 and 29.4 –30.1, each and for GF/C filters wet frozen and GF/C freeze-dried 29.0 –30.7 and 29.4 –32.3, each. There were no significant differences between treatments and for mcyB identical results were observed (see Table 1).
All strains showed comparable cell numbers (1×106 cells ml−1 − 7×107 cells ml−1) during harvesting. With both extraction techniques DNA >12 kb in size was visible after ethidium bromide staining and electrophoresis on 1% agarose gels in 0.5×Tris-borate-EDTA buffer. For Microcystis sp. the standard DNA extraction procedure had Ct-values that were significantly lower (23.6 – 30.9) when compared with Ct-values obtained from the DNeasy Plant system extraction (26.3 – 32.5) (t-test, P < 0.001, n = 10, Fig. 1A). For Planktothrix sp. the difference between the standard DNA extraction (23.6 – 29.2) and the DNeasy Plant system (25.2 – 29.2) was less pronounced albeit significant (t-test, P = 0.006, n = 10, Fig. 1B). For both cyanobacteria the calculated cell numbers were found to be significantly higher in DNA extracts obtained using the standard phenol-chloroform procedure when compared to the DNeasy Plant system extractions (1.6 – 18.8 fold in Microcystis, 0.21-6.2 fold in Planktothrix). The DNeasy Plant system did not reveal improved yields on a per cell basis when cell numbers per extraction column were decreased linearly down to 800 fold (total filter, Ct = 33.2 ± 0.32 (1 SE), ½ filter, Ct = 32.0 ± 0.33, ¼, Ct = 33.5 ± 0.20, 1/10, Ct = 32.6 ± 0.2, 1/100, Ct = 33.9 ± 0.3, 1/200, Ct = 33.1 ± 0.1, 1/400, Ct = 32.4 ± 0.3, 1/800, Ct = 33.0 ± 0.2) and yields compared with the yield extracted using the standard procedure (Ct = 27.2 ± 0.06).
In order to find out whether impurities in the DNA that are inhibitory to the PCR may cause the lower yields obtained by the DNeasy Plant system the following tests with M. flos aquae extracts (showing 18.8 fold lower cell numbers) were performed: (i) DNA obtained by the DNeasy Plant system was spiked with DNA obtained by the standard procedure to balance for the 18.8 fold lower cell number estimate and (ii) the DNA extract obtained by the DNeasy Plant system was 18.8 fold less diluted. Compared with the standard procedure extract (Ct = 29.1 ± 0.02) the spiking treatment (Ct = 29.5 ± 0.51) and the dilution treatment (Ct = 28.6 ± 0.12) showed DNA yields that did not differ significantly.
In order to test the influence of DNA extraction on sensitivity of TNA, DNA extracts obtained through both extraction techniques from filters containing varying cell numbers (102- 108 cells filter−1) then amplified by TNA were compared to cell numbers estimated by microscopy. No significant difference was detectable when comparing microscopic cell counts to cell numbers estimated by TNA from DNA extracts obtained from Microcystis strain HUB53 (Mann Whitney Test, P = 0.86, n = 6) and Planktothrix strain No75 (Mann Whitney Test, P = 0.62, n = 6). Irrespective of the DNA extraction method, cells of both strains were equivalently detected down to a concentration of 105 cells extract−1. Below 105 cells extract−1 the results had greater standard deviation. The lowest detectable signal corresponded to 10 cells per template extracted from 102 cells on a filter.
In Lake Wannsee Planktothrix cell numbers estimated in the microscope varied from 5.9×102 − 1.4×105 ml−1 (Fig. 2). The variation measured by TNA ranged from 4×102 − 1.4×106 ml−1. TNA results on cell number obtained from DNA samples corresponded significantly with cell numbers counted in the microscope. The regression equation was y = −0.08 + 1.07x (R2 = 0.76, n = 20, P < 0.0001), where y is the log10 cell number determined by TNA and x is the log10 cell number counted in the microscope.
The quantitative analyses of water samples require the appropriate storage of samples as well as efficient and reliable DNA extraction. In contrast to the more rigid nature of ME filters the softer GF/C filters do form a distinct pellet after centrifugation subsequent to DNA extraction using phenol-chloroform-isoamyl alcohol. This allows for a distinct separation of the water phase from the phenol-chloroform phase. In contrast the phase separation is disturbed by the rigid pieces of ME filters. Further the results are relevant with regard to the storage and postage of samples that have been collected through the course of recent and past sampling programs. We conclude that freeze-drying of samples gives quantifiable data and that DNA extraction and subsequent TNA of historic samples would be possible. Such a TNA analysis would provide information on toxin genotype composition in populations over longer time periods.
In this study we have found that cell numbers estimated via TNA were highest in extracts obtained by the standard phenol-chloroform procedure. Although cells were harvested during the stationary phase we do not believe that DNA dissolving from dead cells influenced the results obtained. As aliquots of cell harvest were treated identical before the experiment on DNA extraction an influence due to dissolved DNA in the medium during the growth phase seems unlikely. The DNeasy procedure has been developed for DNA extraction from leaves/needles of higher plants containing complex polymers such as cellulose (Qiagen 2003). Because the reduction of cell numbers for DNA extraction down to 1/800 did not reveal higher DNA yields per cell the possibility of over saturation of the DNA extraction columns can be excluded. In addition no impurities that might inhibit the PCR were detected in consequence to spiking the DNeasy kit extracts with DNA obtained by the standard procedure. It is concluded that the lysis and/or extraction efficiency of the DNeasy kit is generally lower. Owing to the variation in polysaccharide composition among cyanobacteria (DePhillips and Vicenzini 1998) and Microcystis sp. (Doers and Parker 1988) and the variation between strains observed in this study we cannot exclude strain specific differences in DNeasy extraction efficiency in field samples.
When testing the sensitivity between both DNA extraction procedures no difference was detectable. The TNA results were predictable down to 105 cells ml−1 implying that if one liter of water is filtered effective cell numbers could be as low as 100 cells ml−1 in order to obtain reliable results. This concentration is below the concentration of 20,000 cells ml−1 that has been considered of relatively mild or low probability of adverse health effects (Falconer et al. 1999). Consequently the DNeasy procedure might be considered an alternative to standard phenol-chloroform DNA extraction for early warning monitoring tasks, particularly to achieve a high throughput in DNA analyses for the health safety analysis of environmental samples. The DNeasy procedure needs only 2 h as opposed to 8 h required for the standard extraction procedure and also replaces cumbersome phenol and chloroform extraction.
The results of this study obtained from field samples from Lake Wannsee demonstrate the application of DNA extraction and subsequent TNA. The overestimation of cell numbers by the TNA during the first year cannot be explained, however, may also be due to errors in sedimentation and/or counting under the microscope. The TNA developed in this study is also expected to provide reliable results applied to samples from other populations of P. agardhii and P. rubescens.
This study was financed by the EU project PEPCY (Toxic and Other Bioactive Peptides in Cyanobacteria, QLK4-CT-2002-02634, www.pepcy.de) and the Austrian Science Funds (P18185).