Sensitivity and amplification efficiency of TNA
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.
Influence of filter types and sample storage on TNA results
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 Ct-values and cell numbers extract−1 (mean ± 1 SE) estimated using TNA (PC, mcyB) from DNA extracts obtained from two different types of filters (GF/C, glass fiber, ME, membrane) and two different types of storage (wet frozen, freeze-dried). (more ...)
Variation in TNA results using two different DNA extraction techniques
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, ). 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, ). 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).
Fig. 1 Cell numbers (mean ± 1 SE) of 10 Microcystis sp. (A) and 10 Planktothrix spp. strains (B) as determined by TNA from DNA extracts obtained either through DNA extraction according to the standard phenol-chloroform procedure (▲) or the DNeasy (more ...)
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.
Application of TNA
In Lake Wannsee Planktothrix cell numbers estimated in the microscope varied from 5.9×102 − 1.4×105 ml−1 (). 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.
Fig. 2 Numbers of Planktothrix cells in Lake Wannsee from June 1999 to October 2000, determined by counting under the inverted microscope (■) or by TNA quantification () of the phycocyanin operon (mean ± 1 SE).