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

Frontiers in Medicine

David Dobnik¹  Maja Ravnikar¹  Maja Štalekar¹  Polona Kogovšek¹  Barbara Jerič Kokelj¹  Miha Arnol²  Sebastian Vencken³  Matjaž Stanonik³ 

[1] Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia;Department of Nephrology, University Medical Centre Ljubljana, Ljubljana, Slovenia;Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia;GenePlanet, Ljubljana, Slovenia;

关键词: kidney transplantation;    droplet digital PCR;    plasma cell-free DNA;    minor allele quantification;    assay evaluation;    graft health monitoring;   

DOI  :  10.3389/fmed.2021.748668

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

CC BY