Merkel Cell Polyomavirus (MCPyV) Detected in Plasma of Post-Bone Marrow Transplant Patients by SYBR Green-Based Real-Time PCR and Melting Curve Analysis
Su S Chen, Jeffrey J Tarrand, Victor Prieto, Pei Lin, Michael H Fernandez, Tooba Hasan, L Jeffrey Medeiros, Carlos Bueso-Ramos. University of Texas MD Anderson Cancer Center, Houston, TX
Background: The novel Merkel cell polyomavirus (MCPyV) was first identified in Merkel cell carcinoma (MCC) in 2008. MCPyV is detected at high frequency (∼80%) in MCC and is also reported in chronic lymphocytic leukemia (CLL). Post-bone marrow transplant (post-BMT) patients are immunosuppressed and frequently have reactivation of normally latent viruses, such as Epstein-Barr virus (EBV) and cytomegalovirus (CMV). There are no reports on detection of MCPyV in post-BMT patients, most likely due to the short time since the first discovery of this virus. Additionally, there are no reports of using plasma samples for detection of MCPyV.
Design: We developed a SYBR Green based real-time PCR and melting curve analysis method to detect MCPyV and we tested plasma samples. We initially developed the method using DNA from a MCPyV-positive cell line (MS-1) and validated the method on clinical samples of MCC (n=5) and CLL (n=2). Subsequently, we tested plasma samples obtained from post-BMT patients that had tested positive for EBV (n=10) or CMV (n=10) as part of routine laboratory workup.
Results: Using MS-1 cell line DNA, MCPyV was detected by SYBR Green-based real time-PCR and melting curve analysis generating a melting temperature peak of 81.7 +/- 0.2 oC. The MCPyV PCR amplicon was confirmed by agarose gel electrophoresis producing the expected amplicon size as well as by DNA sequencing. By serial dilution of MS-1 DNA, the dynamic range and the limit of detection of the real-time PCR assay was established as 5-log10 and 0.16ng DNA, respectively. The analytical performance of the assay was tested on clinical samples with results as follows: 5 of 5 (100%) MCC formalin-fixed paraffin-embedded tissue (FFPET) samples and 1 of 2 CLL peripheral blood samples were positive for MCPyV. Out of 20 post-BMT plasma samples that were previously tested positive for EBV or CMV, MCPyV was detected in 5 (25%): 3 of 10 EBV-positive samples and 2 of 10 CMV-positive samples.
Conclusions: We report a SYBR Green-based real-time PCR and melting curve analysis method for detection of the novel MCPyV and show, for the first time, that this virus can be detected in plasma samples of post-BMT patients. The clinical importance of MCPyV in plasma samples needs further clinical and epidemiological investigation, for which the developed assay can be useful.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 250, Wednesday Afternoon