ISBN 978-3-8439-2516-7

978-3-8439-2516-7, Reihe Mikrosystemtechnik

Naga Deepa Pantulu
Detection of human pathogenic polyomaviruses by a polymer-based DNA biochip platform

169 Seiten, Dissertation Albert-Ludwigs-Universität Freiburg im Breisgau (2015), Softcover, B5

Zusammenfassung / Abstract

Polyomaviruses (PV) belong to the family Polyomaviridae. Human polyomaviruses (HPyVs), namely the JC polyomavirus (JCV) and the BK polyomavirus (BKV), were reported nearly four decades ago, however, their significance in clinical research has tremendously increased with the discovery of ten human polyomaviruses in the past seven years. Our study focused on the detection of pathogenic human polyomavirus species JCV, BKV and MCV based on their T-Ag gene, using a DNA microarray (biochip) based assay.

For this purpose, a DNA biochip is fabricated with virus-specific probes using a simple one-step procedure, briefly a mixture of a photoactive benzophenone group containing copolymer together with the probe sequences is printed on a microarray substrate. Following, brief irradiation, the photopolymer is cross linked and attached to the chip surface and the probes are covalently attached to the polymer network. The two main parameters, the specificity and analytical sensitivity of the DNA chip for the detection of these polyomaviruses are investigated. Finally, in order to validate the results of microarray, comparison with conventional methods including PCR with agarose gel electrophoresis and DNA sequencing is carried out.

Further, our study included investigating the putative role of the polyomaviruses in polyomavirus associated primary and secondary malignancies, here we tested the presence of Merkel cell polyomavirus (MCV) and its mutations in chronic lymphocytic leukemia (CLL) samples. Merkel cell polyomavirus (MCV), the novel human polyomavirus, has been reported in approximately 80% of human Merkel cell carcinomas (MCC). MCC a rare and aggressive skin cancer of neuroendocrine origin, is often linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). Here the presence of MCV in highly purified leukemic cells of 70 chronic lymphocytic leukemia (CLL) patients was analyzed by PCR and DNA sequencing. Subsequently, the samples were analyzed by qPCR, immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) methods. The control population included 82 DNA derived from peripheral blood mononuclear cells (PBMCs) of normal healthy blood donor samples and 24 MCV positive MCC samples.