Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the HHV-6 sequence with the standard and competitor DNA constructs.
- Fig. 2.
(A) Reference curve for TaqMan assay. The dotted lines represent the 95% confidence limits of the assay; the equation of the regression curve obtained by plotting the Ctvalues (y axis) against the indicated amounts of DNA inputs (x axis) is y = 38.534 − 3.495 log(x), with a value of fit (R) equal to 0.999. (B) Results obtained with three reference curves, constructed at different times, to assess reproducibility. In all the resulting equations [curve 1, y = 38.907 − 3.554 log(x); curve 2, y = 38.547 − 3.508 log(x); curve 3, y = 38.147 − 3.422 log (x)], both the slope coefficients and the intercept values were very similar, and no significant difference was found by covariance analysis.
- Fig. 3.
Comparison of assay sensitivity for HHV-6 DNA detection between TaqMan and nested PCR assays. The results are for serial 10-fold dilutions of the reference DNA (from 101 and 106 copies/reaction mixture) and one negative control sample, as detected by TaqMan assay amplification (A) or by nested PCR on an ethidium bromide-stained 2% agarose gel (B). The symbols shown at the top of the nested PCR bands identify the corresponding dilutions of the template. For each dilution, the normalized fluorescence signal (ΔRn) is plotted against the PCR cycle number.
- Fig. 4.
Standard curve for HHV-6 QC-PCR. A fixed amount (103 copies) of the pVU45 calibrator plasmid was amplified in the presence of increasing amounts of the pVU44 standard plasmid (A). Following ethidium bromide staining the relative amounts of amplified products were calculated by densitometric analysis, and ratios between the coamplified products were plotted on a semilogarithmic scale as a function of increasing amounts of the reference DNA (B). The corresponding equation is reported in the upper part of the figure.
- Fig. 5.
Accuracy error of the TaqMan and QC-PCR assays, computed as the average of the measured value after subtraction of the actual value. The error is reported as a function of the actual number of DNA copies to be measured. By both methods, accuracy improved with an increase in the amount of DNA, but the error was systematically lower by the TaqMan assay. In addition, QC-PCR yielded a marked overestimation of the number of copies at all levels.
- Fig. 6.
Detection of HHV-6 subtypes by TaqMan assay. HHV-6AGS (filled columns) and HHV-6B PL1(shaded columns) genome equivalents measured in 1 ml of supernatant derived from cultured PBMCs infected with 0.1 and 10 TCID50s, respectively.
Tables
- Table 1.
Ct values under different PCR conditions
Copy no. Ctvaluea Activation timeb(min) TAQ6a-TAQ6E primer concnc(nM) Probe concnc(nM) Mg2+ concnc(mM) 5 10 15 50-50 50-300 50-900 300-50 300-300 300-900 900-50 900-300 900-900 10 25 50 100 200 2 4 6 7 101 40 40 35 37 35 37 39 35 35 35 38 37 37 36 34 36 34 40 36 34 34 102 40 34 31 38 32 30 33 29 30 33 32 31 34 32 31 32 31 40 31 32 31 103 40 30 28 30 29 28 30 28 28 29 28 28 36 28 28 28 104 40 26 25 25 25 25 25 25 25 25 25 25 27 25 25 25 25 33 24 24 24 105 40 22 21 31 20 20 21 ↵a The data represent the means of three independent experiments.
↵b The optimal AmpliTaq Gold activation time was established by using the manufacturer-suggested PCR conditions.
↵c The different primer, probe, and Mg2+ concentrations were tested by varying a single parameter at a time, using the optimal AmpliTaq Gold activation time (15 min).
- Table 2.
CV of standard plasmid pVU44 quantifications by TaqMan and QC-PCR
Starting DNA copy no. % CV TaqMan assay QC-PCR assay Intra-assay Interassay Intra-assay Interassay 100 197 197 101 67 67 102 30 30 124 274 103 12 14 25 59 104 8 8 36 40 105 7 8 106 5 8 - Table 3.
CV for quantification of HHV-6 in infected tissue samples by TaqMan and QC-PCR assays
Sample no. TaqMan assay QC-PCR assay Quantification (no. of DNA copies) Intra-assay CV (%) Interassay CV (%) Quantification (no. of DNA copies) Intra-assay CV (%) Interassay CV (%) 1 1,585 8 8 1,812 9 15 2 7,553 8 12 12,342 8 17 3 1,280 4 4 1,574 6 11 4 4,300 9 18 7,030 6 19 5 430 24 53 810 31 34
