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Articles |
1
Klinik für Innere Medizin m.S. Hämatologie und Onkologie, Charité-Campus Virchow Klinikum, Humboldt Universität, Augustenburger Platz 1, Forschungshaus 37 R 2.303, D-13353 Berlin, Germany.
2
TIB Molbiol, D-10829 Berlin, Germany.
a Author for correspondence. Fax 49-30-45053929; e-mail andreas.nitsche{at}charite.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments.
Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation.
Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods commonly described to quantify HCMV DNA include competitive PCR assays (6)(7) and hybridization of PCR products to probes in a microwell format. These tests are labor-intensive and require post-PCR handling. Recently, quantitative, fluorescence-based, real-time PCR assays in closed tube systems have been developed. Sequence detection systems commonly used include the exonuclease assay (8) and the hybridization probe assay, which are performed with an ABI PRISM 7700 Sequence Detection System (7700; PE Applied Biosystems) or the LightCycler (Roche Diagnostics) (9), respectively.
The exonuclease probe format is based on the 5'-nuclease activity of
the DNA polymerase that cleaves a hybridized nonextendible exonuclease
probe during the extension phase of the PCR (Fig. 1
) (10). The exonuclease probe is designed to
hybridize specifically to the amplicon and is dual-labeled with a
reporter dye and a quencher dye. The 5'-exonuclease activity of the
polymerase separates reporter dye and quencher dye by hydrolysis of the
exonuclease probe during polymerization (11). Reporter dye
fluorescence increases as it is progressively released from the
proximity of the quencher. This fluorescence is cumulative and
indirectly related to the amount of PCR product (12). An
additional fluorescent dye, 6-carboxy-X-rhodamine (ROX), is added to
serve as an internal passive reference.
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A second, fluorescence-based format for real-time PCR detection is the
hybridization probe format (Fig. 1
). It uses two independent,
single-labeled oligonucleotides that hybridize adjacently on the
amplicon internal to the flanking PCR primers. The upstream
oligonucleotide is labeled at its 3' end with fluorescein (FL) and the
downstream oligonucleotide is labeled at its 5' end with LightCycler
Red 640 (LC Red). Hybridization probes are nonextendible. After
excitation by the light-emitting diode, a fluorescence resonance
energy transfer (FRET) occurs from the FL (donor) to the LC Red
(acceptor), increasing the LC Red signal, which is directly related to
the amount of PCR product before the plateau is reached
(12). In the hybridization probe format, as in the
exonuclease probe format, the hybridization probes may be degraded
during displacement from the amplicon. In contrast to the exonuclease
probe format, the fluorescence signal in the hybridization probe format
is read in the annealing phase and not in the extension phase. However,
both formats allow the quantification of PCR products by measuring the
fluorescence during each cycle. Fluorescence data are used to calculate
the first cycle in which fluorescence exceeds the baseline value by 10
SD of the fluorescence increase during cycles 3–15. This cycle number
is called the threshold cycle (CT). Comparison of
CT values to a calibration curve allows
the absolute quantification of unknown amounts of DNA.
To improve methods for diagnostic quantification of HCMV DNA in the plasma of patients, we studied whether the exonuclease assay could be performed on the LightCycler and compared the results with those obtained on the 7700. Furthermore, we compared the hybridization probe assay with the exonuclease probe assay, both performed on the LightCycler.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pcr
The sequences of the primers, exonuclease probe, and hybridization
probes are given in Table 1
. A schematic representation of the location and orientation of
the oligonucleotides used is given in Fig. 2
.
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All PCR reaction mixtures contained 10x PCR buffer (200 mmol/L Tris-HCl, pH 8.4, 500 mmol/L KCl), 4 mmol/L MgCl2, 0.8 mmol/L dNTP (Life Technologies), 0.8 µmol/L each primer, and 1 U of Platinum Taq DNA polymerase (Life Technologies). The exonuclease assay carried out on the 7700 contained an additional 50 nmol/L exonuclease probe TM2 and 1 µmol/L ROX (Molecular Probes) in a reaction volume of 50 µL. To the exonuclease assay performed on the LightCycler, 50 nmol/L exonuclease probe TM2 and 1.5 g/L bovine serum albumin (BSA; Sigma) were added. The hybridization probe assay contained an additional 150 nmol/L donor hybridization probe, 300 nmol/L acceptor hybridization probe, and 1.5 g/L BSA. Reactions on the LightCycler were performed in 20-µL volumes.
Using the 7700, we performed PCR in 96-well microtiter plates (Perkin-Elmer), whereas glass capillaries were used for PCR in the LightCycler (Roche Diagnostics).
The standard temperature profile for exonuclease assays included an initial denaturation for 3 min at 94 °C, followed by 45 cycles of denaturation at 94 °C for 10 s, annealing at 64 °C for 10 s, and an extension with fluorescence monitoring at 72 °C for 30 s. The standard temperature profile for hybridization probe assays included an initial denaturation for 3 min at 94 °C, followed by 45 cycles of denaturation at 94 °C for 0 s, annealing with fluorescence monitoring at 55 °C for 15 s, and extension at 72 °C for 15 s.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Amplification profiles of both reactions are nearly identical
in the range of 101 to 107
HCMV genome equivalents (Fig. 3
, A and B). There is a good linear
correlation (r >0.995) between the CT
and the logarithm of DNA copy number (Fig. 3C
). All PCRs were performed
in quadruplicate; the intraassay variation (CV) was <5%, and
the interassay variation was <10% for both systems.
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To reduce the assay time, we gradually shortened the individual reaction steps of the PCR program. The most rapid assay that could be carried out on the 7700 without a loss of sensitivity or a decrease in precision consisted of an initial denaturation for 3 min at 94 °C, followed by 45 cycles of denaturation at 94 °C for 5 s, annealing at 64 °C for 15 s, and extension with fluorescence monitoring at 72 °C for 10 s. The minimal resulting total run time was ~80 min. Further shortening of the denaturation step was not possible because time intervals shorter than 5 s cannot be set on the 7700. With the LightCycler, because of its fast ramping, the minimal total reaction time was 20 min, corresponding to initial denaturation for 3 min at 94 °C, followed by 45 cycles of denaturation at 94 °C for 0 s, annealing at 64 °C for 15 s, and extension with fluorescence monitoring at 72 °C for 0 s. Using annealing times <10 s, we observed a loss in sensitivity (CT shift to higher values), annealing times <5 s produced no amplification (data not shown).
hybridization probe reaction
Hybridization probe reactions were carried out exclusively with
the LightCycler, using the reaction conditions given above. To find the
optimum conditions for the detection of HCMV DNA, we tested five
different hybridization probe sets (Fig. 2
). As shown, all of the
hybridization probes hybridized in the same region of the
MIE gene as the exonuclease probe TM2, i.e., between 46 and
98 bp downstream from primer 1F. Two hybridization probe pairs (AD/AA
and BD/BA) hybridized with the sense strand, in a position relatively
close to the primer; the third hybridization probe pair (CD/CA)
hybridized to the antisense strand. Additional hybridization probe
pairs tested were BD/AA and AD/BA. Best results were obtained with
hybridization probe pairs AD/AA and BD/AA (Fig. 4
). There was a linear correlation for both hybridization sets
(r >0.997 for BD/AA; r >0.998 for AD/AA) between
the CT value and the template concentration in a
range between 101 and 107
molecules of the target sequence per assay (Fig. 4C
). All of the
hybridization probe PCR assays were performed in quadruplicate, the
intraassay variation was <5%, the interassay variation was <10%.
There was no difference in total fluorescence yield obtained with
hybridization probe pairs AD/AA and BD/AA, although the distance
between the FL and LC Red is 1 base for set AD/AA and 7 bases for set
BD/AA. Hybridization probe set BD/BA gave a poor correlation between
CT and template concentration. As expected,
hybridization probe set AD/BA yielded very low fluorescence signals
because of an overlap of the two probes for a length of 5 bases.
Hybridization probe set CD/CA did not produce any fluorescence signals
(data not shown). To optimize the fluorescence yield, we varied the
hybridization probe concentration for set AD/AA and BD/AA. As one would
suppose, a decrease of the hybridization probe concentration led to a
loss of sensitivity, whereas a doubling of the hybridization probe
concentration led to an increase in total fluorescence but also to
increased background during the first 15 cycles (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test for sensitivity, precision, reproducibility, and practicality, we compared the 7700 and the LightCycler for the quantification of HCMV DNA. Initially, we were interested to see whether the exonuclease probe format could be transferred from the 7700 to the LightCycler. Subsequently, we compared the results obtained in the exonuclease probe assay with those obtained in the hybridization probe assay.
We have shown that the exonuclease assay can be transferred to the LightCycler without any modifications of the temperature profile or the composition of the reaction mixture except for the addition of low concentrations of BSA to avoid binding of polymerase to the glass capillaries. The exonuclease probe assay enables reliable quantification on both instruments, producing calibration curves with strong correlations as well as low intraassay (<5%) and interassay (<10%) variation.
We chose a detection range of 101 to 107 HCMV genome equivalents/assay, which sufficiently covers the expected values for HCMV DNA load in plasma and peripheral blood leukocytes in immunosuppressed patients [reviewed in Ref. (14)].
Attempts to minimize the assay time by shortening individual reaction steps led to a minimum exonuclease probe assay on the 7700 of 80 min. With the LightCycler, because of its high temperature ramping rates and the good thermal conductivity of the glass capillaries, the exonuclease probe assay was reduced to a total run time of 20 min.
Our second intention was to compare the performance of the exonuclease assay to the hybridization probe assay, both carried out on the LightCycler. We chose three sets of hybridization probes located in the same region as the exonuclease probe and used them in five different pairings. The best results were obtained with hybridization probe sets AD/AA and BD/AA. They showed strong linear correlation between CT value and template concentration. The remaining probe sets produced inferior results. Our data suggest that results obtained with two of the selected hybridization probe sets are comparable to those obtained with the exonuclease probe assay.
According to theoretical considerations, FRET is only effective when energy donor and acceptor molecules are in close proximity, i.e., within a distance of <40 Å. This condition is fulfilled in both hybridization probe sets AD/AA and BD/AA, in which the distances are 1 and 7 bp, respectively. Both sets lead to comparable results. In addition to distance of donor and acceptor molecules, the quality of probe binding to the template is of importance. In the hybridization probe set BD/BA, signal production was inferior to AD/AA and BD/AA, although the gap between the bound probes was 1 bp and therefore fulfilled the theoretical conditions for an effective FRET. This is remarkable because the hybridization probes BA and AA have 16 bases in common and differ only in 6 and 8 nucleotides.
The probe pair AD/BA produced a weak signal that probably is explained by an overlap of both probes over the length of 5 bases. Interestingly, the probe set CD/CA that binds to the antisense strand did not lead to signal production. This phenomenon is as yet unexplained. It may be attributable to the high content of purine bases. We exclude poor probe quality as the reason for this finding because the quality of the fluorescence dye labeling was comparable for all hybridization probes. Moreover, the highest fluorescence dye/DNA ratio was found in hybridization set CD/CA, which produced no fluorescence signal during amplification.
In conclusion, when the exonuclease format is used, both the 7700 and the LightCycler allow quantification of HCMV DNA with the same precision. In the LightCycler, the exonuclease probe can be replaced by a set of hybridization probes without loss in sensitivity or reproducibility of quantification. The two principles of fluorescence signal production (exonuclease probe and hybridization probe) work equally well. However, the question of whether preference should be given to one instrument and/or one fluorescence detection format is as yet unanswered. The 7700 has the advantage of simultaneous analysis of 96 samples compared with only 32 samples in the LightCycler. Additional advantages include easier handling, better suitability to automation, and the ability to excite and detect a large number of fluorescent dyes. In contrast, the LightCycler in our assay system requires a minimum running time of only 20 min compared with 80 min with the 7700. The LightCycler can deal with both the exonuclease and the hybridization probe format, whereas the FRET process cannot be monitored with the 7700 because the long-wavelength acceptor dye LC Red is directly excited by the laser light source. Moreover, for the construction of two hybridization probes, more suitable target DNA sequences must be available than for the construction of one exonuclease probe. In contrast, the synthesis of a dual-labeled exonuclease probe is technically more demanding than the synthesis of two single-labeled hybridization probes.
Altogether, the 7700 appears to be useful for the processing of large numbers of sample under standard conditions, whereas the LightCycler has its strength in smaller sample numbers and the use of various reaction conditions.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 1991;88:7276-7280.
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