Rapid, equipment-free extraction of DNA from skin biopsies for point-of-care diagnostics.

TitleRapid, equipment-free extraction of DNA from skin biopsies for point-of-care diagnostics.
Publication TypeJournal Article
Year of Publication2024
AuthorsManning JCade, Boza JManuel, Cesarman E, Erickson D
JournalSci Rep
Volume14
Issue1
Pagination13782
Date Published2024 Jun 14
ISSN2045-2322
KeywordsBiopsy, DNA, Viral, Herpesvirus 8, Human, Humans, Molecular Diagnostic Techniques, Nucleic Acid Amplification Techniques, Point-of-Care Systems, Sarcoma, Kaposi, Skin
Abstract

Kaposi's sarcoma (KS) is a cancer affecting skin and internal organs for which the Kaposi's sarcoma associated herpesvirus (KSHV) is a necessary cause. Previous work has pursued KS diagnosis by quantifying KSHV DNA in skin biopsies using a point-of-care (POC) device which performs quantitative loop-mediated isothermal amplification (LAMP). These previous studies revealed that extracting DNA from patient biopsies was the rate limiting step in an otherwise rapid process. In this study, a simplified, POC-compatible alkaline DNA extraction, ColdSHOT, was optimized for 0.75 mm human skin punch biopsies. The optimized ColdSHOT extraction consistently produced 40,000+ copies of DNA per 5 µl reaction from 3 mg samples-a yield comparable to standard spin column extractions-within 1 h without significant equipment. The DNA yield was estimated sufficient for KSHV detection from KS-positive patient biopsies, and the LAMP assay was not affected by non-target tissue in the unpurified samples. Furthermore, the yields achieved via ColdSHOT were robust to sample storage in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer prior to DNA extraction, and the DNA sample was stable after extraction. The results presented in this study indicate that the ColdSHOT DNA extraction could be implemented to simplify and accelerate the LAMP-based diagnosis of Kaposi's sarcoma using submillimeter biopsy samples.

DOI10.1038/s41598-024-64533-3
Alternate JournalSci Rep
PubMed ID38877073
PubMed Central IDPMC11178891
Grant ListU01CA269199 / / U.S. National Institutes of Health /
Related Faculty: 
Ethel Cesarman, M.D., Ph.D.

Pathology & Laboratory Medicine 1300 York Avenue New York, NY 10065 Phone: (212) 746-6464
Surgical Pathology: (212) 746-2700