Journal
ELSEVIER
of Virological
Methods
47 (1994) 189-202
Journal of Virological ~~t~o~l~
Quanti~cation of cytom~galovirus DNA in blood specimens from bone marrow transplant recipients by the polymerase chain reaction B.K. Rawal*@, J.C. Bootha,
S. Fernandoa,
P.D. Butcher”,
R.L. Powlesb
aDepnrtment of Medical Microbiology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK, h Leukaemia Unit, The Royal Marsden Hospital. Downs Road. Sutton, Surrey, SM2 SPT, UK (Accepted
3
November 1993)
Abstract
A nested PCR system for cytomegalovirus (CMV) DNA in blood specimens from bone marrow transplant recipients is described, in which the biotinylated tritium-labelled product from the second round of PCR is quantified using streptavidin-coated fluorometric Scintillation Proximity Assay (SPA) beads (Amersham, UK). This assay has been compared with a PCR procedure based on limiting-dilution, in which the end-point is determined visually following electrophoresis in agarose gel. The two systems were shown to be equivalent in sensitivity and specificity on testing stored serial blood samples from six CMV antibodypositive allogeneic bone marrow transplant patients who developed viraemia as detected by conventional methods of virus isolation in tissue culture. Key
words: Cytomegalovirus; Quantitative plant recipient; Peripheral blood leukocyte
polymerase chain reaction; Bone marrow trans-
1. Introduction is a serious compIication of allogeneic treatment with ganciclovir and high-titred immunoglobulin has reduced the mortality rate from 80% to about 40%, but either treatment alone offers little benefit (Winston et al., 1990). Early treatment, prefer-
Cytom~galovirus (CMV) bone marrow transplantation.
‘Corresponding
author.
pneumonitis Combined
Fax: f 44 081 6821320.
0166-0934/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0 166-0934(93)EOlSO-2
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ably before the onset of pneumonitis, is important but requires sensitive and rapid surveillance techniques for early evidence of CMV infection. Traditional methods of diagnosis have the disadvantages of low sensitivity and delay between testing and obtaining a positive result. Amongst procedures that are relatively non-invasive, the detection of viraemia has been shown to carry the highest predictive value for impending CMV disease (Meyers et al., 1990) therefore blood specimens from at-risk patients are screened routinely by various techniques. The PCR is one method being evaluated widely for diagnosing CMV-related illness (Einsele et al., 1991a; Brytting et al., 1991) and for monitoring the effectiveness of therapy (Einsele et al., 1991b). Moreover, using this technique, there is increasing interest in establishing the clinical relevance and prognostic implications of quantifying the amount of CMV present in a particular clinical sample. Various methods have been investigated for this, including measuring the amount of PCR-product DNA, performing limiting dilution endpoint titration of the target DNA and competitive assays in which the target (patient’s) DNA is co-amplified with a known amount of a ‘standard’ DNA fragment, usually in the form of a plasmid, which is identical to the target DNA sequence apart from containing a distinguishing deletion or insertion or a unique restriction site (Gerdes et al., 1993; Fox et al., 1992). This enables the PCR products for the virus and the standard to be differentiated on the basis of size, hybridization to an insert-specific probe, or susceptibility to cleavage by the appropriate restriction enzyme. The above procedures have been evaluated almost exclusively with single-round PCR techniques. However, the development of quantitative nested PCR would offer the advantages of greater specificity as well as sensitivity. Our own experience of single-round PCR on DNA extracted from the peripheral blood leukocytes (PBL) of bone marrow transplant recipients is that the amount of product DNA is so small as to be seldom detected visually following electrophoresis in agarose gels and staining with ethidium bromide; however, unequivocal results are obtained by nested PCR. Our interest in a nested PCR procedure that gives quantitative results has led us to examine two methods. One is a simple semi-quantitative nested PCR in which the levels of viral DNA in the sample under test are determined using a limitingdilution approach in which the end-point is read visually, following agarose gel electrophoresis. The other uses a radioactive detection system for measuring concentrations of PCR product DNA (Quant-Amp, Amersham, UK). In this, one of the PCR primers is labelled with biotin and, in addition, tritiated dTTP is included in the PCR reaction mixture. Thus the PCR product DNA is end-labelled with biotin and is labelled throughout with tritium. On completion of the amplification cycles, an aliquot of the final reaction mixture is removed and added to streptavidin-coated fluomicrospheres (SPA beads - Amersham, UK). The capture of the biotinylated product DNA by the streptavidin brings the tritium close enough to the microsphere so that the fluor incorporated within it is excited to emit a pulse of light that is detectable in a scintillation counter (see Fig. 1). This system has been applied to an established nested CMV DNA PCR, previously optimised in this laboratory (Fernando et al., 1993), which uses primers to the morphological transformation region II (mtr II) of the CMV genome (Khan et al., 1991). The results of both PCR meth-
B.K. Rawal et al./Journal of Virological Methods 47 (19941 189-202
191
O_-‘-_‘* B -----_-_ *
+
PCR with biotinytated primer (8) and tritiated dllP
(*)
Free unincorporated tritiated dlTP is too far away from the SPA beads to activate them.
cJ=====**:* Streptavidin-coated SPA bead
Radiolabelled biotinylated PCR product is captured by Streptavidin-coated SPA beads resulting in emission of light measured in scintillation counter.
Fig. 1. Principle of scintillation proximity assay (SPA) as applied to PCR.
ods have been compared one with another and with those of conventional and rapid methods of virus isolation (Steel et al., 1988) and serology.
2. Materials and methods 2.1. DNA extraction Patients undergoing allogeneic bone marrow transplantation were routinely followed up for CMV surveillance on a weekly basis. After inoculation of cell cultures for conventional and rapid virus isolation, buffy coat samples were mixed with an equal volume of the cryopreservative ‘glycigel’ (10 mM NaCl, 0.5 mM EDTA, 39% glycerol (v/v), 1.5% gelatin (w/v), 0.1% sodium azide (w/v)) and stored at -20°C for up to 3 months (Kaye et al., 1991). Ninety samples from six patients were tested retrospectively. These patients were all CMV antibody-positive pre-transplant and had all developed CMV viraemia at some point post-transplant. The buffy coat/ ‘glycigel’ mixture was rapidly thawed in a 37°C water bath and 1 ml was transferred to a screw-capped 1.6 ml microfuge tube and the cells pelleted at 14000 r-pm for 1 min. The red cells in the pellet were lysed by washing 3 times in lysis buffer (0.32 M sucrose, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl*, 1% Triton X-100 (v/v)). DNA was extracted from the resulting pelleted cell nuclei in 100 ~1 of extraction buffer (50 mM KCI, 10 mM Tris-HCI, pH 8.3, 2.5 mM MgC12, 0.01% gelatin (w/v), 0.45% NP40 (v/v), 0.45% Tween 20 (v/v)), to which proteinase K (Sigma, UK) was freshly added to a final concentration of 60 pg/ml. This mixture was incubated at 60°C for 2 h followed by boiling for 5 min to inactivate the proteinase K. The DNA concentra-
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B.K. Rawal et al./Joumal of Virological Methods 47 (1994) 189-202
tion in each sample was measured (prior to boiling) using a TKO-100 minifluorimeter (Hoefer Scientific Instruments, San Francisco, CA). The DNA extracts were diluted in sterile distilled water to give 100 ng DNA in 10 ~1, which was the volume added to 40 ~1 of the first-round PCR mix. In addition, 3 further IO-fold dilutions of DNA were made so that nested PCR was carried out using 100 ng, 10 ng, 1 ng or 0.1 ng of buffy coat DNA. 2.2. Primers Four primers derived from the mtr II gene were used (Khan et al., 1991). The external pair, yielding a 234 bp product, were P5 (5’ CTGTCGGTGATGGTCTCTTC 3’) and P6 (5’ CCCGACACGCGGAAAAGAAA 3’); the internal pair, yielding a 168 bp product, were P7 (5’ TCTCTGGTCCTGATCGTCTT 3’) and P8 (5’ GTGACCTACCAACGTAGGTT 3’). In the radioactive quantitative studies, 5’biotinylated primer P8 was used. Primers to the histidyl tRNA synthetase gene (Taylor-Wiedman et al., 1991) yielding a 360 bp product were Tl (5’ CTTCAGGGAGAGCGCGTGCG 3’) and T2 (5’ TCATCAGGACCCAGCTGTGC 3’). These were applied to 100 ng (in 10 ~1) of all the DNA samples (in a non-nested PCR) to ensure the presence of amplifiable human genomic DNA in the extracts, and the absence of significant inhibitors of the PCR. 2.3. Conditions for
carrying
out the PCR
DNA extraction, PCR master-mix preparation and PCR product analysis were carried out in separate rooms with dedicated pipettes and racks. Racks were washed in 1 M HCl after each use. Sterile filter-plugged disposable pipette tips were used to avoid cross contamination. The reaction conditions and cycle parameters had been optimised previously in this laboratory and were identical for the histidyl tRNA synthetase PCR and for each round of the nested mtr II PCR. Positive control DNA (extracted from tissue culture cells infected with the laboratory strain AD169) and negative controls (DNA extracted from a CMV antibody-negative person’s blood and PCR reaction mix without added template) were included in each run. The PCR reaction mix (final volume 50 ~1) consisted of 1 PM each primer, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgC12, 0.001% gelatin, 0.2 mM each deoxynucleotide triphosphate, and 2.5 U cloned Taq polymerase. The samples were covered with two drops of mineral oil and after 5 min at 94°C were amplified through 30 cycles as follows: 30 s at 94°C 30 s at 57°C and 90 s at 72°C ending with a final extension step for 7 min at 72°C. When proceeding to the nested PCR, the tubes from the first-round reaction were spun at 14000 rpm for 2 min and then placed under a short wave (254 nm) UV lamp for 5 min (to eliminate surface contamination), prior to removing 2 ~1 which was transferred to tubes containing fresh master-mix but with the inside primers. After amplification as before, 10 ~1 was removed, mixed with 15 ~1 of tracking dye and electrophoresed through a 1.5% agarose gel (type II: Medium EEO, Sigma, UK) containing ethidium bromide (0.5 pg/ml) at 100 volts. The gel was examined and photographed; bands of the
B.K. Rawal et aLlJournal
of Vimlogical
expected size were identified by comparison PhiX174, RF DNA; Gibco-BRL).
Methods 47 (1994) 189-202
with DNA
markers
193
(Hue111 digest of
2.4. Radioactive quantitative PCR Reagents were kindly given by Amersham, UK, in the form of ‘Quant-Amp’ kits and [methyl,1’,2’-3H]thymidine 5’-triphosphate, 90-130 Ci/mmol assay ([3H]dTTP). The first-round PCR, using 100 ng of the patient’s DNA, was as described above. However, for the nested reaction, the tubes contained biotinylated primer P8 and the nucleotide mix included 1 &i tritiated dTTP. To prepare this, the t3H]dTTP (150 &i in 250 ~1) was first desiccated in a centrifugal concentrator with the exhaust from the vacuum pump being vented into a fume hood; the dried label was reconstituted in 75 ~1 cold dNTP mix (from the ‘Quant- Amp’ kit), then 675 ~1 sterile water was added to give a final volume of 750 ~1 of which 5 ~1 was used per PCR reaction. 2 ~1 of the first-round PCR product was transferred to three second-round tubes which were cycled as before. On completion of the nested PCR, 25 ~1 aliquots of each sample were removed, placed in plastic beta scintillation vials followed by 500 ~1 diluted SPA bead suspension (0.5 mg/ml); after gentle agitation, the vials were counted for 1 min each in a scintillation counter (LKB RackBeta II). The mean of the 3 replicate counts was calculated for each sample. For background controls, 3 reagent blank vials were counted which contained only 500 ~1 SPA beads and 25 ~1 water. Also included in each PCR run, in parallel with the nested reaction, were a set of standards consisting of four lOO-fold dilutions of purified CMV-specific DNA of known concentration (this had been prepared from AD169 DNA by PCR-amplification with primers P5 and P6), the first dilution providing 100 pg of template in 2 ~1. Each dilution was amplified in the secondround PCR in triplicate. A standard curve was plotted of mean counts per minute (cpm) vs. amount of template and this was used for determining the amount of PCR-product DNA which was present after the first-round amplification for each of the samples tested.
3. Results 3.1. Detection of CMV DNA in the PBL of healthy seropositives Previous experience in this laboratory has shown that the nested PCR procedure gives positive results (on average, 1 out of 3 tests) on DNA from healthy CMV antibody-positive adults when 1 pg of PBL DNA is added to the first-round reaction mixture (Fernando et al., 1993). However, such large amounts of PBL DNA were difficult to recover from bone marrow transplant patients, because of leukopenia, particularly in the weeks immediately following the transplant. The maximum amount of DNA (in 10 ~1) available reliably for testing was 100 ng; testing this from 11 healthy CMV antibody-positive adults gave negative results by both methods (data not shown).
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B.K. Rawal et al./.Journal of Virological Methods 47 (1994) 189-202
1 2 34
5 6 7 8 9101? 12
Fig. 2. Sensitivity for the detection of CMV DNA by the limiting dilution method. Lanes 1 and 12: Hoe111 digest of PhiX174 molecular weight markers. Lanes 2-9: lo-fold dilutions of purified first-round PCR product starting with 1 ng in lane 2. Lane 10: positive control (AD169 DNA). Lane 11: negative control (sterile water).
3.2. Detection
limits of the PCR methods
Serial IO-fold dilutions of purified DNA, in the form of first-round PCR product which had been generated from the DNA of tissue culture cells infected with the AD169 laboratory strain of CMV were prepared. Each dilution was subjected to the appropriate second-round (nested) PCR procedure for the limiting-dilution method and for the radioactive quantitative method. For the limiting-dilution method, the products of the nested PCR were electrophoresed in agarose gels and the end-point was read as the highest dilution of the starting DNA which resulted in a visible band (Fig. 2). For the radioactive quantitative method, the products of the nested PCR reaction were counted for radioactivity then a graph was plotted of mean cpm vs. amount of specific target DNA added to the reaction. The end-point in this case was determined as the highest dilution (lowest DNA concentration) which gave a mean cpm greater by 50% than the mean cpm for the background control (Fig. 3). By both methods, the smallest amount of the target DNA detected by the second-round PCR was 10 fg; this was equivalent to 4 x lo4 molecules. 3.3. Retrospective recipients
testing
of serial blood specimens
from
bone marrow
Serial specimens of heparinised blood (90 in all) from 6 bone marrow were available, stored frozen in ‘glycigel’ (see Materials and Methods).
transplant
recipients DNA ex-
B.K. Rawal et al./Journal
blank Amount
of Virological Methods
-2 of
first
0 round
2 template
195
47 (1994) 169-202
4
6 added
6 (log
Fig. 3. Sensitivity for the detection of CMV DNA by the radioactive quantitative derived cut-off of 50% greater than the reagent blank mean cpm is shown.
fg) PCR. The arbitrarily
tracted from them was tested by both PCR procedures, each patient’s samples being processed together as a single batch. In the limiting-dilution method, each patient’s DNA was tested in amounts of 100 ng, 10 ng, 1 ng and 0.1 ng by the first-round PCR, then 2 ~1 of each of these reaction mixtures was reprocessed in the secondround PCR, the products being examined for visible specific bands after agarose gel electrophoresis. By the radioactive quantitative method, the first-round PCR was performed only on 100 ng of patients’ DNA; thereafter the second-round PCR was performed in triplicate on 2 ~1 of the first-round product, using the biotinylated primer and [3H]dTTP, and the amount of second-round product was measured using the SPA beads. All the patients’ samples were successfully amplified in a single-round PCR with the primers to the histidyl tRNA synthetase gene, the reaction being carried out on 100 ng of the sample DNA (data not shown). Every time a batch of PCR tests was put up, known CMV-positive and -negative DNA preparations were processed in parallel, as controls. In the radioactive quantitative method, a range of concentrations of the same standard DNA preparation as used in the experiment in Fig. 3 was processed in the second-round PCR, in order to generate a standard curve from which the amount of target DNA corresponding to a particular mean cpm reading could be read off. Fig. 4a shows the standard curves obtained in 6 tests carried out on different occasions; Fig. 4b shows the mean for all 6 curves, the coefficient of variation about each point ranging from 9.&29.6%. The results obtained using both of the PCR methods to test the serial specimens
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B.K. Rawal et al./Journal
of Virological Methods 47 (1994) 189-202
2000 -
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6
(log
fg)
for the radioactive quantitative PCR: (a) curves obtained on titrating in six different experiments; (b) mean valueskone S.D. as calculated
the same from the
from the six patients are shown in Fig. 5 (A-F), together with the results of tests carried out on the buffy coat specimens at the time of routine follow-up to detect infective virus in tissue culture and the details of the duration of anti-CMV therapy.
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The two PCR procedures showed remarkable concordance in the detection of dynamic changes in the level of CMV DNA with time after transplant. The limitingdilution method detected changes in the level of CMV DNA over a range of 2--3logi0, whereas the radioactive quantitative method detected changes over a 34logio scale. The latter was potentially more accurate than the limiting-dilution method, being capable of detecting differences of less than IO-fold which was the minimum in the limiting-dilution method. Precipitate changes in the levels of CMV DNA with time were detected using the PCR, mainly in association with episodes of treatment with ganciclovir or foscarnet. Patients B, D and E showed a single episode of CMV viraemia by the PCR. This was tirst detected at 4-6 weeks post-transplant, resolved on treatment with ganciclovir and remained at below the base-line until the last time of testing, at 15-24 weeks post-transplant. Patients A and C showed two episodes, the first beginning at 4 weeks and 6 weeks, the second at 12 weeks and 17 weeks, respectively; in both cases the earliest peak of CMV DNA disappeared following the administration of ganciclovir and reappeared following the discontinuation of the therapy. Patient F showed a more complex picture in that high levels of CMV DNA were detected in the first specimen tested, at 4 weeks post-transplant; these resolved following treatment with foscarnet, then reappeared and again resolved following the giving of ganciclovir, but thereafter they recurred and persisted at moderate levels up to the last time of testing at 19 weeks post-t~nsplant. If each episode of viraemia is regarded as a separate entity, the PCR procedures provided a positive result one week earlier than virus detection in tissue culture in patients A, B and C. On the other hand, virus culture was as effective as PCR in heralding the first episode of CMV infection in patients C, D, E and F. Viraemia was detected in patient B only by the rapid method, whereas, in all the other patients their first isolation-positive sample was positive by rapid and conventional virus isolation in tissue culture. Virus culture tended to be positive in specimens containing particularly high levels of CMV DNA, however many such specimens were negative for infectious virus. In some instances this was probably because the PCR was detecting non-replicating viral DNA in patients exposed to antiviral therapy. On the other hand, virus isolation in tissue culture appeared to be an inconsistent marker of high virus load in the circulation, even at times when the patient was not on ganciclovir (see patients C and F). Only patients B and F were symptomatic at the time of CMV viraemia: patient B complained of a dry cough and patient F had pyrexia, chest pain and dyspnoea which resolved after treatment. Stored serum samples (collected at the same time points as the heparinised blood
Fig. 5. Post-transplant follow-up of bone amount (ng) of patient’s total DNA found od. Solid squares: amount of CMV DNA quantity of first-round PCR product DNA viraemia as detected by conventional and/or foscarnet.
marrow allograft recipients A-F. Open squares: minimum to be positive for CMV DNA by the limiting-dilution methpresent in 100 ng of patient’s total DNA as reflected in the (logfg) detected using the radioactive quantitative method. v: rapid virus isolation in tissue culture. Gan: ganciclovir. Fos:
B.K. Rawal et al.lJournal of Virological Methods 47 (1994) 189-202
199
samples) were tested also by EIA for antibodies to CMV. All were negative for CMV IgM except for two from patient C; these were collected at weeks 3 and 12 post-transplant, that is, before the appearance of each peak of CMV DNA. CMV IgG levels were strongly positive in all patients throughout the period of investigation and showed only minor variation, none of which had any relevance to the PCR results nor to the results of virus isolation (data not shown).
4. Discussion Limiting-dilution PCR is a technically simple procedure which detects the presence or absence of specific target DNA sequence directly in serial dilutions of the sample under test. It is best suited for detecting wide variation in the amounts of a target DNA sequence in different specimens such as occurs, with time, after the treatment of CMV viraemic patients with antivirals. Accuracy depends on the number of replicate samples that are tested at each dilution interval, four or five being required for resolving differences of less than 0.5 loglo. Such numbers make the test very expensive for routine use. By comparison, the novel radioactive quantitative system for detecting PCR product DNA is intrinsically more accurate, fewer samples being needed to give a result that is more reliable for detecting smaller differences between samples than the limiting-dilution method. Also, the fact that the radioactive method gives a numerical result allows the possibility of statistical analysis. In the present study the potential value of the limiting-dilution method was dependent on major changes occurring in the levels of CMV DNA with time after infection (during weekly follow-up) and major changes following treatment with ganciclovir. The successful application of the SPA bead-based detection system is dependent on the amount of product DNA after the first-round PCR being proportional to the amount of CMV target DNA in the patient’s sample and also on the DNA extraction procedure not inducing any inhibitory activity for the PCR. The latter appeared not to be the case as judged by the lack of inhibition when the extracts were amplified for the histidyl tRNA synthetase gene; the amount of target DNA added to the PCR being no greater than 100 ng may also have helped. That the radioactive quantitative method gave a valid measure of the amount of CMV target DNA was shown by the close concordance between the results of this and the limiting-dilution method. The pattern of results in both tests was remarkably similar, showing that the amount of first-round product DNA in the PCR was proportional to the level of specific target DNA in the sample as revealed by limitingdilution. Our data indicate that quantitative PCR could be reliably applied to post-transplant CMV surveillance of bone marrow recipients by using either of the two methods described here. Neither method appears to yield false negatives or false positives. The upper limit of target DNA applied to the first round PCR was kept to 100 ng in both methods to eliminate the possibility of detecting latent CMV and to take into account the fact that before engraftment, the buffy coat samples from bone marrow patients do not generally yield more than 10-100 pg/ml DNA by the meth-
od of extraction used here. When the conventional nested PCR was originally evaluated in this laboratory, healthy CMV antibody-positive individuals occasionally gave positive results when tested using 1 pg DNA in the first-round PCR, presumably due to the detection of latent viral DNA. The 11 healthy CMV antibodypositive adults studied here were negative by both PCR assays when tested at 100 ng. An important advantage of the radioactive quantitative system is that it can be applied to aid optimisation of the conditions for any PCR, as in this study, given its comparability with the limiting-dilution method. Increasing the specific activity of the tritium label or using more than one radiolabelled dNTP would theoretically improve the sensitivity of this system. The limiting-dilution PCR required each positive sample to be tested in at least eight reactions (100 ng, 10 ng, 1 ng, 0.1 ng, each being nested) whilst the radioactive method required each positive sample to be tested in only four reactions (i.e. a single tube containing 100 ng of the patient’s DNA in the first round followed by triplicate testing in the second round with label). The second round triplicate counts per minute were generally very close, so that duplicate testing may be considered, to lower the cost. However, even allowing for the cost of extra Tuq polymerase and dNTPs required for the limiting-dilution method, the radioactive quantitative method is significantly more expensive to perform. In addition, facilities suitable for the handling and disposal of tritium are required as well as a scintillation counter. This inevitably adds to the time taken to perform the assay, especially if a microtitre plate format for scintillation counting is not available. Compared to the limiting-dilution method, in which the specificity of the PCR product DNA can be judged according to its molecular size and homogeneity on agarose-gel electrophoresis, checks on the specificity of the radioactive assay require using standard non-biotinylated primers with tritiated dTTP to generate labelled PCR product, then probing this in solution with a 5’-biotinylated oligonucleotide complementary to a specific internal sequence of the product DNA; scintillation counting would then be carried out after adding the SPA beads, as in the format described in this study. This would obviate the need for Southern blotting. Novel assays that are designed to quantify PCR products using non-radioactive technology have also been described (Vliegler et al., 1992) which would be interesting to compare with the radioactive system described here. The clinical relevance of the quantification of viruses in specimen material varies depending upon the nature of the condition and the frequency of testing (Fox and Emery, 1992; Saltzman et al., 1992). For CMV surveillance in bone marrow transplant recipients at weekly intervals, the observed fluctuations in the level of CMV DNA with time after transplant and in response to therapy are sufficiently dramatic to be easily detected by the relatively insensitive limiting-dilution technique. More frequent investigation, at closer intervals, searching for more subtle changes in viral load, would be possible using the radioactive quantitative PCR. The present study was undertaken retrospectively, with all the samples from any one patient being tested simultaneously. Had the testing been done prospectively, this would have imposed even greater constraints on the quantitative PCR which have not been considered here. These include the need to ensure comparable sensi-
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201
tivity in tests performed on different days with the same batches of reagents and, in due course, with different batches of reagents. As demonstrated in the present study, the radioactive quantitative PCR, because of its numerical response, is well suited for constructing standard curves with control preparations of specific DNA, which can then be applied to overcoming these problems.
5. Acknowledgements The authors are indebted to Amersham, UK for generously providing reagents and to the staff of the CMV reference laboratory, St. George’s Hospital Medical School (Yvonne Tryhorn, Andy Shipp and Fiona Sydney) for the results of routine tests on patients’ specimens. B.K.R. is supported by the Leukaemia Research Fund.
6. References Bitsch, A., Kirchner, H., Dupke, R. and Bein, G. (1993) Cytomegalovirus transcripts in peripheral blood leukocytes of actively infected transplant patients detected by reverse transcription-polymerase chain reaction. J. Infect. Dis. 167, 74&743. Brytting, M., Sundqvist, V.-A., Stalhandske, P., Linde, A. and Wahren, B. (1991) Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides. J. Viral. Methods 32, 1277138. Einsele, H., Steidle, M., Vallbracht, A., Saal, G.J., Ehninger, G. and Muller, A.C. (1991a) Early occurrence of human cytomegalovirus infection after bone marrow transplantation as demonstrated by the polymerase chain reaction technique. Blood 77, 11041110. Einsele, H., Ehninger, G., Steidle, M., Vallbracht, A., Muller, M., Schmidt, H., Saal, J.G., Wailer, H.D. and Muller, CA. (1991 b) Polymerase chain reaction to evaluate aniviral therapy for cytomegalovirus disease. Lancet 338, 1170-I 172. Fernando, S., Booth, J., Boriskin, Y., Butcher, P., Steel, H., Tryhorn, Y., Corbishley, C., Keeling, P., Murday, A. and McKenna, W. (1993) The association of cytomegalovirus infection with posttransplantation cardiac rejection as studied using the polymerase chain reaction. J. Med. Viral. (in press). Fox, J.C. and Emery, V.C. (1992) Quantification of viruses in clinical samples. Rev. Med. Virol. 2, 195203. Fox, J.C., Grifftths, P.D. and Emery, V.C. (1992) A method to quantify human cytomegalovirus DNA using the polymerase chain reaction J. Gen. Virol. 73, 240552408. Gerdes, J.C., Spees, E.K., Fitting, K., Hiraki, J., Sheehan, M., Duda, D., Jarvi, T., Roehl, C. and Robertson, A.D. (1993) Prospective study utilizing a quantitative polymerase chain reaction for detection of cytomegalovirus DNA in the blood of renal transplant patients, Transplant. Proc. 25, 1411~1413. Kaye, S., Loveday, C. and Tedder, R.S. (1991) Storage and preservation of whole blood samples for use in detection of human immunodeficiency virus type-l by the polymerase chain reaction. J. Virol. Methods 35, 217-226. Khan, G., Kangro, H.O., Coates, P.J. and Heath, R.B. (1991) Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44, 360-365. Meyers, J.D., Ljungman, P. and Fisher, L.D. (1990) Cytomegalovirus excretion as a predictor of cytomegalovirus disease after marrow transplantation: importance of cytomegalovirus viremia. J. Infect. Dis. 162, 3733380. Salzman, R.L., Quirk, M.R. and Jordan, M.C. (1992) High levels of circulating cytomegalovirus DNA reflect visceral organ disease in viremic immunosuppressed patients other than marrow recipients. J.
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47 (19Y4)
189-202
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