Abstract
Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further. DNA was quantified using two detection methods: qPCR and digital PCR (dPCR). Using six punches with the Qiagen Investigator kit gave the best overall DNA yield compared with other extraction methods. Including three punches, however, gave better DNA extraction efficiency. Reference material could be detected using qPCR and dPCR in DBS spiked with 5000 copies/mL of blood (approximately 15 copies per 3 mm of punch). The optimal DNA extraction protocol was used on DBS samples from a custom recombinant adeno-associated virus administration study and showed successful detection of vector targets in DBS samples.
DNA extraction methods for dried blood spots (DBS) were evaluated to determine the suitability of the use of DBS as an alternative matrix for transgene detection towards gene doping screening. DNA was quantified using two detection methods: qPCR and dPCR. Using the Qiagen Investigator kit for DNA extraction with six DBS punches gave a better recovery of targets than three. However, three punches gave better extraction efficiency. DNA can be detected down to 5000 copies/mL of blood using qPCR and dPCR. image