Virology

Real-Time Quantitative PCR for Human Herpesvirus 6 DNA

Giuseppe Locatelli, Fabio Santoro, Fabrizio Veglia, Alberto Gobbi, Paolo Lusso, Mauro S. Malnati
DOI: 

Article Figures & Data

Figures

  • Fig. 1.
    Fig. 1.

    Schematic representation of the HHV-6 sequence with the standard and competitor DNA constructs.

  • Fig. 2.
    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 Ct values (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.
    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.
    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.
    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.
    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.Ct valuea
    Activation timeb(min) TAQ6a-TAQ6E primer concnc(nM)Probe concnc(nM)Mg2+ concnc(mM)
    5101550-5050-30050-900300-50300-300300-900900-50900-300900-9001025501002002467
    101 404035373537393535353837373634363440363434
    102 403431383230332930333231343231323140313231
    103 40302830292830282829282836282828
    104 402625252525252525252525272525252533242424
    105 40222131202021

    • ↵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 assayQC-PCR assay
    Intra-assayInterassayIntra-assayInterassay
    100 197197
    101 6767
    102 3030124274
    103 12142559
    104 883640
    105 78
    106 58
  • Table 3.

    CV for quantification of HHV-6 in infected tissue samples by TaqMan and QC-PCR assays

    Sample no.TaqMan assayQC-PCR assay
    Quantification (no. of DNA copies)Intra-assay CV (%)Interassay CV (%)Quantification (no. of DNA copies)Intra-assay CV (%)Interassay CV (%)
    11,585881,812915
    27,55381212,342817
    31,280441,574611
    44,3009187,030619
    543024538103134
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