Comparison of Quantitative Real-Time PCR and Digital PCR to Detect the Polyomavirus in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) prevalence in Merkel cell carcinoma (MCC) cases is controversial. The detection and quantification of MCPyV DNA is mainly performed by PCR techniques using formalin-fixed, paraffin-embedded (FFPE) tissues. The aim of this study is to compare the performance of two different molecular techniques, specifically the quantitative Real-Time PCR (qPCR) and digital PCR (dPCR). Samples from 31 cases of MCC excisional surgical biopsies were analyzed. DNA extraction and purification from clinical samples were performed using the QIAcube Qiagen automated nucleic acid extractor. After the extraction, MCPyV was detected by qPCR and dPCR using specially designed primers and probes. Of the 31 MCC samples under study, the MCPyV genome was detected in 11 samples (35%) by qPCR compared with 20 samples (65%) detected by dPCR. Notably, 65% of primary tumors were positive for MCPyV (15/23). The viral genome was detected in 75% of tumors located at UV-exposed sites (6/8), 55% of tumors at partially UV-protected sites (5/9), and 67% of tumors at UV-protected sites (4/6). Our results showed a better sensitivity of dPCR in detecting the MCPyV genome in MCC samples compared with traditional qPCR techniques.

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

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.