Droplet‐digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females. Issue 3 (23rd September 2019)

• Record Type: Journal Article
• Authors: Tagliapietra, Andrea; Rotondo, John Charles; Bononi, Ilaria; Mazzoni, Elisa; Magagnoli, Federica; Gonzalez, Lucia Oton; Contini, Carlo; Vesce, Fortunato; Tognon, Mauro; Martini, Fernanda
• Is Part Of: Journal of cellular physiology. Volume 235:Issue 3(2020:Mar.)
• Page Start: 1888
• Page End: 1894
• Publication Date: 2019-09-23
• Subjects: abortion -- chorionic villi -- ddPCR -- MCPyV -- PBMC; Physiology -- Periodicals; Cell physiology -- Periodicals; 571.6
• DOI: 10.1002/jcp.29213
• Languages: English
• ISSNs: 0021-9541

Abstract

Abstract: Droplet‐digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)‐affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females ( n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [ SD ]) copy/10 4 cells in SA and 3.02 ( ± 1.86 [ SD ]) copy/10 4 cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [ SD ]) copy/10 4 cells and 4.09 ( ± 4.26 [ SD ]) copy/10 4 cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA‐ and 2/5 (40%) VI‐MCPyV‐positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples. Abstract : Droplet‐digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)‐affected females. This ddPCR assay showed high accuracy, sensitivity and specificity in detecting MCPyV DNA in clinical samples. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA‐ and 2/5 (40%)