Rosaria Arvia1, Mauro Sollai2, Federica Pierucci3, Carmelo Urso4, Daniela Massi2, Krystyna Zakrzewska5. 1. Department of Experimental and Clinical Medicine, University of Florence, Italy. 2. Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Italy. 3. Department of Experimental and Clinical Biomedical Sciences 'Mario Serio', Molecular and Applied Biology Research Unit, University of Florence, Florence, Italy. 4. Department of Anatomic Pathology, Dermatopathology Section-S. M. Annunziata Hospital, AUSL Toscana Centro, Florence, Italy. 5. Department of Experimental and Clinical Medicine, University of Florence, Italy. Electronic address: krystyna.zakrzewska@unifi.it.
Abstract
BACKGROUND: Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. OBJECTIVE: To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. METHODS: Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. RESULTS: The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥105 copies per reaction, while the upper limit of ddPCR was 104 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). CONCLUSIONS: The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome.
BACKGROUND:Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. OBJECTIVE: To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. METHODS: Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. RESULTS: The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥105 copies per reaction, while the upper limit of ddPCR was 104 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). CONCLUSIONS: The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome.
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