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Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients

Authors :
Barbara Jerič Kokelj
Maja Štalekar
Sebastian Vencken
David Dobnik
Polona Kogovšek
Matjaž Stanonik
Miha Arnol
Maja Ravnikar
Source :
Frontiers in Medicine, Vol 8 (2021)
Publication Year :
2021
Publisher :
Frontiers Media S.A., 2021.

Abstract

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patients' plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA.

Details

Language :
English
ISSN :
2296858X
Volume :
8
Database :
Directory of Open Access Journals
Journal :
Frontiers in Medicine
Publication Type :
Academic Journal
Accession number :
edsdoj.271cc744d59341db92a1eb2f1f004fd6
Document Type :
article
Full Text :
https://doi.org/10.3389/fmed.2021.748668