DETECTing Merkel Cell Polyomavirus in Merkel Tumors
| Autor: | Sudhir Krishna, Anjali Vijaykumar, Reety Arora, Komal Gupta |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
DNA damage Merkel cell polyomavirus Biochemistry Genetics and Molecular Biology (miscellaneous) Biochemistry Merkel cell polyomavirus (MCPyV) 03 medical and health sciences Merkel cell carcinoma 0302 clinical medicine molecular diagnosis medicine CRISPR Molecular Biosciences lcsh:QH301-705.5 Molecular Biology DETECTR Polymerase Original Research Merkel tumors Reporter gene Cas12a biology business.industry CRISPR diagnosis biology.organism_classification medicine.disease 3. Good health 030104 developmental biology medicine.anatomical_structure lcsh:Biology (General) 030220 oncology & carcinogenesis Cancer research biology.protein Skin cancer business Merkel cell circulatory and respiratory physiology |
| Zdroj: | Frontiers in Molecular Biosciences Frontiers in Molecular Biosciences, Vol 7 (2020) |
| ISSN: | 2296-889X |
| DOI: | 10.3389/fmolb.2020.00010 |
| Popis: | Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post-integration (~80% cases), or by UV-mediated DNA damage. Interestingly, overall survival of MCV-positive Merkel cell carcinoma patients is better, making this differential information of significant diagnostic and prognostic value. Also, MCV provides a direct target for therapy in MCC patients. Currently, the methods used for diagnosis of MCV in tumors are often discordant and unreliable. Here we used a guided molecular scissors based–DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV-positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme–gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that MCV DETECTR system can detect MCV integrated in Merkel tumor rapidly, specifically and with femto-molar sensitivity. Our study is a preliminary, proof-of-principle analysis showing the use of CRISPR for MCV diagnosis. Further validation in human tumor samples is needed for its clinical use in the near future. This new system is promising and we hope it can be coupled with immunohistochemical studies to diagnose the viral status of MCC in clinics soon. |
| Databáze: | OpenAIRE |
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