Here we report how variability in the human cytomegalovirus genome sequence may seriously affect the outcome of its molecular diagnosis. A real-time quantitative PCR assay targeting the major immediate-early gene failed to detect the viral load in some, but not all, clinical samples from hematooncological patients. By amplification and sequencing the DNA across the regions targeted by this assay we found a number of nucleotide substitutions which accounted for decreased primer/probe binding. This decreased the sensitivity of the assay up to 1,000-fold.

Center of Molecular Biology and Gene Therapy Department of Internal Medicine Hematooncology University Hospital Brno Brno Czech Republic

J Clin Microbiol. 2008 Apr;46(4):1571-2. doi: 10.1128/JCM.02473-07. PubMed

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Boeckh, M., B. Fries, and W. G. Nichols. 2004. Recent advances in the prevention of CMV infection and disease after hematopoietic stem cell transplantation. Pediatr. Transplant. 8(Suppl. 5):19-27. PubMed

Boeckh, M., M. Huang, J. Ferrenberg, T. Stevens-Ayers, L. Stensland, W. G. Nichols, and L. Corey. 2004. Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J. Clin. Microbiol. 42:1142-1148. PubMed PMC

Brytting, M., J. Wahlberg, J. Lundeberg, B. Wahren, M. Uhlen, and V. A. Sundqvist. 1992. Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing. J. Clin. Microbiol. 30:955-960. PubMed PMC

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. PubMed PMC

Drago, L., A. Lombardi, E. De Vecchi, G. Giuliani, R. Bartolone, and M. R. Gismondo. 2004. Comparison of nested PCR and real time PCR of herpesvirus infections of central nervous system in HIV patients. BMC Infect. Dis. 4:55. PubMed PMC

Gault, E., Y. Michel, A. Dehee, C. Belabani, J. C. Nicolas, and A. Garbarg-Chenon. 2001. Quantification of human cytomegalovirus DNA by real-time PCR. J. Clin. Microbiol. 39:772-775. PubMed PMC

Griscelli, F., M. Barrois, S. Chauvin, S. Lastere, D. Bellet, and J. H. Bourhis. 2001. Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR. J. Clin. Microbiol. 39:4362-4369. PubMed PMC

Herrmann, B., V. C. Larsson, C. J. Rubin, F. Sund, B. M. Eriksson, J. Arvidson, Z. Yun, K. Bondeson, and J. Blomberg. 2004. Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus. J. Clin. Microbiol. 42:1909-1914. PubMed PMC

Kearns, A. M., M. Guiver, V. James, and J. King. 2001. Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus. J. Virol. Methods 95:121-131. PubMed

Leruez-Ville, M., M. Ouachee, R. Delarue, A. S. Sauget, S. Blanche, A. Buzyn, and C. Rouzioux. 2003. Monitoring cytomegalovirus infection in adult and pediatric bone marrow transplant recipients by a real-time PCR assay performed with blood plasma. J. Clin. Microbiol. 41:2040-2046. PubMed PMC

Li, H., J. S. Dummer, W. R. Estes, S. Meng, P. F. Wright, and Y. W. Tang. 2003. Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J. Clin. Microbiol. 41:187-191. PubMed PMC

Nitsche, A., N. Steuer, C. A. Schmidt, O. Landt, and W. Siegert. 1999. Different real-time PCR formats compared for the quantitative detection of human cytomegalovirus DNA. Clin. Chem. 45:1932-1937. PubMed

Nye, M. B., A. R. Leman, M. E. Meyer, M. A. Menegus, and P. G. Rothberg. 2005. Sequence diversity in the glycoprotein B gene complicates real-time PCR assays for detection and quantification of cytomegalovirus. J. Clin. Microbiol. 43:4968-4971. PubMed PMC

Pignatelli, S., P. Dal Monte, G. Rossini, and M. P. Landini. 2004. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev. Med. Virol. 14:383-410. PubMed

Prentice, H. G., and P. Kho. 1997. Clinical strategies for the management of cytomegalovirus infection and disease in allogeneic bone marrow transplant. Bone Marrow Transplant. 19:135-142. PubMed

Retiere, C., B. M. Imbert, G. David, P. Courcoux, and M. M. Hallet. 1998. A polymorphism in the major immediate-early gene delineates groups among cytomegalovirus clinical isolates. Virus Res. 57:43-51. PubMed

Schaade, L., P. Kockelkorn, K. Ritter, and M. Kleines. 2001. 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. J. Clin. Microbiol. 39:3809. PubMed PMC

Stocher, M., V. Leb, M. Bozic, H. H. Kessler, G. Halwachs-Baumann, O. Landt, H. Stekel, and J. Berg. 2003. Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run. J. Clin. Virol. 26:85-93. PubMed

Tanaka, N., H. Kimura, K. Iida, Y. Saito, I. Tsuge, A. Yoshimi, T. Matsuyama, and T. Morishima. 2000. Quantitative analysis of cytomegalovirus load using a real-time PCR assay. J. Med. Virol. 60:455-462. PubMed

Zaia, J. A. 2002. Prevention and management of CMV-related problems after hematopoietic stem cell transplantation. Bone Marrow Transplant. 29:633-638. PubMed

Zweygberg Wirgart, B., M. Brytting, A. Linde, B. Wahren, and L. Grillner. 1998. Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations. J. Clin. Microbiol. 36:3662-3669. PubMed PMC

In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

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