C. pneumoniae-infected HeLa cells were Proteinase K-treated and a serial four-fold dilution series was prepared with concentrations near and past the detection limit for both PCR methods used. These reference samples were tested with a real-time PCR and a conventional PCR both specific for C. pneumoniae (see table 1 for sample volumes). The results obtained from the reference samples with the two PCR methods are shown in table 2. Samples were blinded prior to analysis, to avoid experimentator-induced bias. Both PCR methods correctly identified the five first samples in the dilution series, and no false positives were observed. In the conventional PCR (method 2) it is an advantage that a higher template volume can be used, which might make it possible to detect lower C. pneumoniae concentrations. This is not possible for the real-time PCR method (method 1) as the capillaries limit the reaction volume to 20 μl. In conclusion, the two PCR methods had the same limit of detection. We avoided using inclusion-forming units (IFU) as a measure of sensitivity because it is probably not very informative as different culture systems may vary considerably and because the determination procedure, which involves cultivation and counting under a microscope, can be operator dependent [8]. Measuring DNA concentration with a spectrometer is less operator dependent and is therefore probably easier to compare between laboratories. Furthermore, IFU only measures the number of viable EB in a preparation, it does not account for RB and dead EB, which also contain genomic DNA amplifiable by PCR.
When performing quantitative real-time PCR, it is assumed that the PCR efficiency of the standard samples is the same as that of the unknown samples. This is the basis for the calculations made. Therefore, it is important to determine the efficiency in both standards samples and unknown samples. PCR efficiency depends e.g. on inhibition in the sample, how well primers and probes are designed, and how well the PCR conditions are optimised. PCR efficiency was determined in the reference samples described above and in standard samples. The standard samples were a 10-fold dilution series of purified genomic C. pneumoniae DNA with known concentrations. Threshold cycles obtained with the real-time PCR for duplicates of the reference and standard samples were plotted against log to the concentration of the samples (log to the concentration: arbitrary numbers with four-fold difference for the reference samples, as concentrations were unknown) (fig. 1). Two straight lines were drawn and the slopes were determined by linear regression to be -3.848 (standard error = 0.259) for the reference samples and -3.144 (standard error = 0.065) for the standard samples. The two slopes were found to be significantly different (z = 2.64, P = 0.008). Efficiency is derived from the idealized function for the amount of PCR product formed: N = N0 En, where N is number of amplified molecules, N0 is the initial number of molecules, n is the number of amplification cycles and E is the efficiency which is ideally 2. The standard curves are derived from the function described above: n = -(1/log E) * log N0 + (log N/log E). Therefore, the slope of the line equals -(1/ log E) and the efficiency can be calculated from the slope. From the slopes the efficiency of the real-time PCR in the reference samples was determined to be 1.82 and in the standard samples to be 2.08. The fact that the efficiency is a little larger than 2 is probably caused by the linear regression analysis which does not take into account that the efficiency can maximally be 2. Because of the difference in efficiency between reference and standard samples an inaccuracy in the determined concentration of the reference samples is present. As the efficiency in the reference samples is lower, the concentration determined on basis of the standard samples will be an underestimation. This is seen in table 2, as it would be highly unlikely to be able to determine the lowest concentration 0.05 copies/μl if it was not an underestimation caused by different efficiencies. The underestimation factor can be estimated for each cycle number. At cycle number 30 and with the described difference in efficiency the concentration would be underestimated 23-fold (see fig. 2). Differences in efficiency of PCR can be caused by several factors e.g. inhibitors and storage of the sample, but different batches of primers, probes and enzymes might also influence the efficiency. The DNA for the reference samples was released by Proteinase K treatment only with no subsequent purification; therefore inhibitors might have been present. Furthermore, as a larger volume of reference sample (6.8 μl) than standard sample (2 μl) was used, this could increase inhibition. On the other hand, the amplification efficiency appeared to be identical for all dilutions as indicated by the regression line, so inhibition from the sample preparation might not be the explanation; the reference samples had been stored in polypropylene tubes for more than a month before analysis, and as DNA binds to polypropylene [12], this could also affect the efficiency observed as this effect alters the DNA concentrations especially in the dilute samples [12].
example four fold serial dilution practice
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Determination of the efficiency for the standard, reference and clinical samples. The threshold cycle is the cycle number at which the fluorescence curve for a given sample crosses the user-defined noise band. Log to the concentration for the reference samples is set as log to arbitrary numbers with four-fold difference as the reference samples are four-fold serial dilutions. The actual concentration is not needed when determining the efficiency as it depends on the slope of the line only.
In table 4 slopes and standard errors for dilution series of negative samples spiked with 103 copies/μl C. pneumoniae DNA and the two samples (samples no. 1 and 60) with low C. pneumoniae DNA concentration are shown. Two-fold dilution series were prepared for each sample. The slopes for the spiked samples were compared to the slope of the standard samples from the same run. P-values for a t-test testing whether slopes differ significantly are shown. Three (sample no's. 101, 131 and 160) of the 20 samples have slopes significantly different from the slope of the corresponding standard and none of these were C. pneumoniae positive when not spiked. This means that the efficiency differs significantly and quantification of these samples is not accurate. It should be noted though, that for the samples 1, 60 and 133 (the three C. pneumoniae positive samples) there are no signs of inhibition. Therefore, quantification of the positive samples is probably reasonably accurate.
C. pneumoniae EB was purified by gradient centrifugation as previously described [16] and genomic DNA was purified with the Genomic-tip system from Qiagen following instructions from the manufacturer. DNA concentration and purity were analysed by agarose gel electrophoresis and measurement of OD at 260 and 280 nm. A ten-fold standard dilution series of purified C. pneumoniae genomic DNA with known concentration was used for quantification, concentrations ranged between 105 and 1 copies/μl [9]. The Lightcycler software determined the threshold cycles for the standard samples and a standard curve was generated. The threshold cycle for unknown samples was determined for two replicates and concentrations were calculated from the standard curve also using the Lightcycler software. A negative control containing all reagents except template DNA was included in all runs. A sample was considered to be positive when the fluorescence curve was sigmoidal and reached fluorescence levels above the fluorescence threshold set in the Ligthcycler software during analysis.
C. pneumoniae was cultivated in HeLa cells as previously described [20]. Cells were scraped off with a rubber policeman, centrifuged and resuspended in TE-buffer. Proteinase K was added and the mixture was incubated for one hour at 55C. Proteinase K was inactivated by incubation at 95C for 10 min. A serial four-fold dilution series was prepared in TE-buffer with 10 dilutions ensuring a wide range of concentrations. Samples with C. pneumoniae DNA concentrations near the detection limit should therefore be present for the methods tested. In addition, two negative controls containing TE-buffer only were included. The two PCR methods were tested on the reference samples and their relative sensitivities were obtained.
The real-time PCR was used for determination of PCR efficiency in a selection of clinical samples in the following way. For negative samples and samples with low C. pneumoniae DNA concentration spiking with 103 copies/μl C. pneumoniae was done. Afterwards a 2-fold dilution series with 8 dilutions was done in water with 10 μg/ml yeast RNA as carrier nucleic acid [9]. For each of the dilutions we measured threshold cycles and plotted them towards log to relative concentrations of the dilutions. The slope was determined and compared to the slope of the standard samples from the same run.
A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M ... Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration curves with a logarithmic scale. A tenfold dilution for each step is called a logarithmic dilution or log-dilution, a 3.16-fold (100.5-fold) dilution is called a half-logarithmic dilution or half-log dilution, and a 1.78-fold (100.25-fold) dilution is called a quarter-logarithmic dilution or quarter-log dilution. Serial dilutions are widely used in experimental sciences, including biochemistry, pharmacology, microbiology, and physics. 2ff7e9595c
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