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Journal of Clinical Microbiology, October 2001, p. 3809-3809, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3809.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
LETTERS TO THE EDITOR
Detection of Cytomegalovirus DNA in Human Specimens by
LightCycler PCR: Melting Point Analysis Is Mandatory To Detect Virus
Strains with Point Mutations in the Target Sequence of the
Hybridization Probes
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LETTER |
In our recently published paper we described the application of
the LightCycler technology for the detection of cytomegalovirus (CMV)
DNA in human plasma and urine (1). We are now able to give
further information gained since September 2000, when this PCR
procedure was introduced as the standard assay for the detection of CMV
DNA in our diagnostic laboratory.
As of June 2001, we had tested 912 specimens using this assay.
According to the described test protocol, 185 specimens (20.3%) were
positive for CMV DNA. A melting point (Tm) of
59.2°C was observed for the hybridization probes with PCR amplicons
of all positive specimens. Another 12 specimens (1.3%) were negative in quantitative analysis but generated a significant peak, with a
Tm of 53.1°C, in the melting point analysis.
Gel electrophoresis revealed a distinct PCR product of about 250 bp
with these specimens that corresponds to the targeted 254-bp amplicon.
DNA sequencing of the PCR products with the decreased melting point
confirmed the specific amplification of CMV DNA using these specimens
as a template. The decline of the melting point is caused by a point mutation in position 630 of the CMV glycoprotein B gene (GenBank accession no. A13758), resulting in a shift from cytosine to thymidine.
This strain variant is not included in the databases and causes a
mismatch with position 5 of the LC-Red 640-labeled acceptor fluorophore
probe. However, the existence of further strain variations in the
amplified region cannot be excluded.
The melting point analysis allows the reliable detection of CMV strain
variants harboring this point mutation. Specificity of the signal
should be further confirmed by gel electrophoresis. Melting point
analysis is mandatory to detect virus strain variants in the target
sequence of the hybridization probes with the described LightCycler CMV
PCR assay.
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FOOTNOTES |
*
Phone: 49 241 8088460 Fax: 49 241 8082483 E-mail: mkleines{at}post.klinikum.rwth-aachen.de
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REFERENCES |
| 1.
|
Schaade, L.,
P. Kockelkorn,
K. Ritter, and M. Kleines.
2000.
Detection of cytomegalovirus DNA in human specimens by LightCycler PCR.
J. Clin. Microbiol.
38:4006-4009[Abstract/Free Full Text].
|
| | | | |
Lars Schaade
Peter Kockelkorn
Klaus Ritter
Michael Kleines*
Division of Virology
Department of Medical Microbiology
University Hospital
RWTH Aachen
D-52057 Aachen, Germany
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Journal of Clinical Microbiology, October 2001, p. 3809-3809, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3809.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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