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Quantitation of cytomegalovirus (hCMV) DNA and β-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients

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Abstract

Even now rare human cytomegalovirus (hCMV) reactivation is still a life-threatening complication after solid organ transplantation. Although PCR techniques are regarded as the most sensitive detection methods for hCMV, their accuracy and reproducibility are limited. This is a major disadvantage with quantitative PCR assays, which are thought to provide valuable information about hCMV latency or active viral replication in transplant patients. To enhance the diagnostic safety of quantitative hCMV PCR, we developed a duplex real-time fluorescence PCR that is capable of quantifying hCMV DNA and β-actin DNA as internal control simultaneously within one reaction. By the use of 6-carboxyfluorescein and hexa-chloro-6-carboxyfluorescein as reporter fluorophores and 4-(4′-dimethylamino-phenylazo) benzoic acid as dark quencher dye, hCMV DNA and β-actin DNA could be quantified in parallel in a wide linear range from 101 to 107 copies, each. To test the clinical applicability of this approach, we investigated hCMV DNA kinetics in peripheral leukocytes of three hCMV antigen-positive and four antigen-negative patients after liver transplantation, as assessed by intracellular hCMV pp65 alkaline phosphate-anti-alkaline phosphate (APAAP) complex. While all APAAP-negative individuals remained PCR negative, kinetics of HCMV DNA in leukocyte DNA samples of APAAP-positive patients correlated closely with hCMV antigen tests. Here, comparison of separate and simultaneous target quantitation revealed identical results. It is of interest that, while single hCMV antigen positivity is commonly not regarded as a reliable parameter of viral reactivation, in our study a viral load greater than 104 copies/2×105 β-actin DNA copies clearly indicated a subsequent increase in APAAP-positive leukocytes. We conclude that with the presented method the reliability of hCMV quantitation via real-time PCR can be substantially increased and may be used to monitor hCMV kinetics in vivo.

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Authors and Affiliations

  1. Department of Medicine, Division of Hematology and Oncology, Charité Campus Virchow-Klinikum, Humboldt University Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

    Joachim Hänfler, Karl Anton Kreuzer, Katja Laurisch, Christian Andreas Schmidt & Helmut Oettle

  2. Department of General Surgery and Transplantation Surgery, Charité Campus Virchow-Klinikum, Humboldt University Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

    Nada Rayes & Peter Neuhaus

Authors
  1. Joachim Hänfler
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  2. Karl Anton Kreuzer
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  3. Katja Laurisch
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  4. Nada Rayes
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  5. Peter Neuhaus
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  6. Christian Andreas Schmidt
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  7. Helmut Oettle
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Correspondence to Joachim Hänfler.

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Hänfler, J., Kreuzer, K.A., Laurisch, K. et al. Quantitation of cytomegalovirus (hCMV) DNA and β-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients. Med Microbiol Immunol 192, 197–204 (2003). https://doi.org/10.1007/s00430-002-0166-6

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  • DOI: https://doi.org/10.1007/s00430-002-0166-6

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