Previous Article | Next Article 
Journal of Clinical Microbiology, May 1999, p. 1658-1659, Vol. 37, No. 5
0095-1137/99/$04.00+0
LETTERS TO THE EDITOR
Evaluation of PCR Primers for Cytomegalovirus Detection
 |
LETTER |
We read with great interest the article by Mendez et al.
(2) published in a recent issue evaluating different PCR
primers for cytomegalovirus (CMV) detection. As CMV PCR becomes a
frequently used assay in the management of immunocompromised patients,
such as transplant recipients and human immunodeficiency virus-infected individuals, an increasing number of laboratories are establishing in-house CMV PCR assays. In designing a PCR assay, the choices of
specimen, sample preparation method, primers, probes, and amplification and detection conditions play a critical role in the sensitivity and
specificity of the assay. Mendez et al. (2) compared primer pairs for HindIII-X fragment, EcoRI fragment D,
immediate-early antigen 1 gene (IEA1), and major immediate-early gene
(MIE) and found sensitivites of 94, 87, 32, and 20%, respectively.
Prompted by their paper, we evaluated the HindIII-X
primer pair versus EcoRI fragment D (4) and MIE
2783-3114 (1), both of which are routinely used in our
laboratory. For this comparison, we used 10 plasma samples containing a
low level of CMV DNA (<1,000 copies/ml). DNA was extracted from each
plasma sample by using four protocols: protocols A and B involved DNA
extraction from 5 and 10 µl of plasma, respectively, with Chelex
resin (Bio-Rad); protocol C started with 300 µl of plasma, which was
centrifuged at 24,000 × g for 1 h after which DNA was
extracted from the pellet with Chelex resin; and in protocol D, plasma
samples were heated at 100°C for 50 to 55 s. The amounts of
template DNA used in each PCR tube in protocols A, B, C, and D
corresponded to 1, 2, 60, and 2 µl of plasma, respectively. The
amplification was performed as previously described (3), by
using 1 U of Pyrococcus furiosus (Pfu) DNA
polymerase (Stratagene) in appropriate buffer, 50 µM each
deoxynucleoside triphosphate (dNTP), and 0.5 µM each primer in
50-µl reaction volumes. PCR products were separated according to
their molecular weight by agarose gel electrophoresis, stained with
ethidium bromide, and visualized under UV light. Positive and negative
controls were included in each run. In contrast to Mendez et al., we
found a sensitivity of 78% for the EcoRI primers, which was
superior to the MIE and HindIII primers' sensitivities of
18 and 5%, respectively. All the specimens in which CMV was detected
by HindIII primers also had CMV detected by EcoRI
and MIE primers. The differences between primer pair sensitivities were
stable across sample preparation protocols. Protocol D, which used 2 µl of heat-inactivated plasma sample/reaction tube, yielded the
highest number of positive results across primer pairs, whereas protocol A, Chelex extraction of DNA from 10 µl of plasma, had the
lowest yield. There are several differences between our assay and that
described by Mendez et al. which might account for the discrepant
results, mainly the utilization of different specimens, plasma versus
peripheral-blood leukocytes (PBL), and different DNA polymerases,
buffers, dNTP and primer concentrations, and cycling conditions. In
addition, for this comparison we did not perform a Southern blot,
although the assay used for selecting the 10 specimens relied on
Southern blot confirmation. These discrepancies emphasize the
importance of performing extensive evaluations of in-house PCR methods
and adapting assay conditions to the primers used and the DNA template
area that is interrogated.
| |
REFERENCES |
| 1.
|
Chou, S.
1992.
Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region.
J. Clin. Microbiol.
30:2307-2310[Abstract/Free Full Text].
|
| 2.
|
Mendez, J. C.,
M. J. Espy,
T. F. Smith,
J. A. Wilson, and C. V. Paya.
1998.
Evaluation of PCR primers for early diagnosis of cytomegalovirus infection following liver transplantation.
J. Clin. Microbiol.
36:526-530[Abstract/Free Full Text].
|
| 3.
|
Weinberg, A.,
D. Spiers,
G. Cai,
C. Long,
R. Sun, and V. Tevere.
1998.
Evaluation of a commercial PCR kit for diagnosis of cytomegalovirus infection of the central nervous system.
J. Clin. Microbiol.
36:3382-3384[Abstract/Free Full Text].
|
| 4.
|
Wolf, D. G., and S. A. Spector.
1992.
Diagnosis of human cytomegalovirus central nervous system disease in AIDS patients by DNA amplification from cerebrospinal fluid.
J. Infect. Dis.
166:1412-1415[Medline].
|
| | | | |
Adriana Weinberg
Shaobing Li
University of Colorado Health Sciences Center
4200 East Ninth Ave.
Denver, Colorado 80262
|
| |
AUTHORS' REPLY |
Optimization and standardization of molecular diagnostic laboratory
assays for the detection of CMV infection in organ transplantation have
been important goals at our institution for effective management of
these patients. Using primers directed to the EcoRI fragment D region of CMV, infection was detected a mean of 13 days before the
onset of symptomatic infection with high sensitivity (92%) and
specificity (100%), but the method lacked specificity (57%) for
predicting disease due to this virus (5). Based on these results, a strategy for implementation of preemptive therapy of CMV
infection in liver transplant recipients was performed by using the
EcoRI primers, once more demonstrating their high
sensitivity for the detection of early CMV infection. However, we had
to abandon these primers for clinical use because isopsoralen
incorporation into the small, 152-bp amplicon was not effective for the
control of carryover contamination (2, 4, 6).
Using the specimens collected in such a prospective and interrupted
preemptive-therapy trial, we sought primers selected by computer
analysis of the MIE gene. In practice, these primers failed to detect
CMV DNA early in the posttransplantation period. These results prompted
us to evaluate several primer sets for early detection of CMV DNA which
were described by other investigators and successfully implemented into
their transplant programs; this was the basis for the published study
to which Weinberg and Li refer.
Weinberg and Li compared results with only two of the four primer sets
that we evaluated (406 bp [1] and 152 bp
[6]). CMV primers directed to the MIE gene were
different in the two studies (332 bp in the study by Weinberg and Li
and 370 bp in our study). In addition, Weinberg and Li cited different
conditions for almost every variable in the PCR protocol (specimen
type, nucleic acid extraction method, DNA polymerase enzyme, etc.); nevertheless, the sensitivities for detection of CMV DNA with primers
to the EcoRI fragment D gene were surprisingly similar in
the two studies (87% in our study and 78% in that of Weinberg and
Li). Other variables that need to be highlighted are the type and
number of samples and method of sample collection, that is, plasma
versus PBL (a few plasma samples [10 samples] in the study of
Weinberg and Li versus many PBL samples [148 samples] in our study)
and preselected stored samples versus prospectively collected and
stored PBL specimens from a much larger number of individuals (21 individuals in our study). These factors likely account for the
observed differences in the sensitivities of primers for detecting CMV DNA.
We believe that valid comparison of PCR results between laboratories
requires use of the same experimental conditions, as an absolute
prerequisite, to expect results that can be accurately evaluated. In
our study, we obtained equivalent results using our standard PCR
protocol versus conditions customized in our laboratory that complied
exactly with those described by investigators such as Drouet et al. for
the primers directed to the HindIII-X locus (1).
Moreover, we have continued to use HindIII-X locus primer
sets in our ongoing trial demonstrating, once again, high sensitivity
and specificity.
Clinical management of organ transplant patients, AIDS patients, and
other immunocompromised populations susceptible to CMV infections is
extremely complex. We agree with Weinberg and Li that in-house PCR
protocols need to be carefully standardized to accurately detect and
quantitate CMV DNA, at least for use in these local patient populations
(3). Most importantly, several commercial systems are being
developed and evaluated for the qualitative and quantitative detection
of CMV DNA or RNA. In the future, implementation of these tests will
allow for accurate and meaningful comparisons of data among laboratories.
| |
REFERENCES |
| 1.
|
Drouet, E.,
S. Michelson,
G. Denoyel, and R. Colimon.
1993.
Polymerase chain reaction detection of human cytomegalovirus in over 2000 blood specimens correlated with virus isolation and related to urinary virus excretion.
J. Virol. Methods
45:259-276[Medline].
|
| 2.
|
Espy, M. J.,
T. F. Smith, and D. H. Persing.
1993.
Dependence of polymerase chain reaction product inactivation protocols on amplicon length and sequence composition.
J. Clin. Microbiol.
31:2361-2365[Abstract/Free Full Text].
|
| 3.
|
Mendez, J.,
M. Espy,
T. F. Smith,
J. Wilson,
R. Wiesner, and C. V. Paya.
1998.
Clinical significance of viral load in the diagnosis of cytomegalovirus disease after liver transplantation.
Transplantation
65:1477-1481[Medline].
|
| 4.
|
Patel, R.,
T. F. Smith,
M. Espy,
D. Portela,
R. H. Wiesner,
R. A. F. Krom, and C. V. Paya.
1995.
A prospective comparison of molecular diagnostic techniques for the early detection of cytomegalovirus in liver transplant recipients.
J. Infect. Dis.
171:1010-1014[Medline].
|
| 5.
|
Patel, R.,
T. F. Smith,
M. Espy,
R. H. Wiesner,
R. A. F. Krom,
D. Portela, and C. V. Paya.
1994.
Detection of cytomegalovirus DNA in sera of liver transplant recipients.
J. Clin. Microbiol.
32:1431-1434[Abstract/Free Full Text].
|
| 6.
|
Spector, S. A., and D. G. Wolf.
1993.
Early diagnosis of human cytomegalovirus disease in transplant recipients by DNA amplification in plasma.
Transplantation
56:330-334[Medline].
|
| | | | |
Thomas F. Smith
Mark J. Espy
Carlos V. Paya
Division of Clinical Microbiology
Division of Infectious Diseases
Mayo Clinic
Rochester, Minnesota 55905
|
Journal of Clinical Microbiology, May 1999, p. 1658-1659, Vol. 37, No. 5
0095-1137/99/$04.00+0
This article has been cited by other articles:
-
Weinberg, A., Schissel, D., Giller, R.
(2002). Molecular Methods for Cytomegalovirus Surveillance in Bone Marrow Transplant Recipients. J. Clin. Microbiol.
40: 4203-4206
[Abstract]
[Full Text]
Top
Letter
References
