Detection of adeno‐associated viral DNA in equine post‐administration frozen blood and plasma samples after long‐term storage

• Published in: Drug testing and analysis 2024-05, Vol.16 (5), p.498-503
• Main Authors: Maniego, Jillian, Pesko, Bogumila, Habershon‐Butcher, Jocelyn, Hincks, Pamela, Taylor, Polly, Stewart, Graham, Proudman, Christopher, Ryder, Edward
• Format: Article
• Language: English
• Subjects: AAV; Animals; Blood tests; Dependovirus - genetics; DNA, Viral - blood; Doping in Sports; Drug testing; gene doping; Genetic testing; Genetic Vectors; Green Fluorescent Proteins - genetics; Horse sports; Horses - blood; massively parallel sequencing; Plasma; Plasma - chemistry; Plasma - virology; qPCR; Time Factors; transgenes
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3569

Gene doping in horses is a threat to the fairness in sport and has serious implications for animal welfare. To investigate the effect of long‐term storage on the detection of AAV in plasma and whole blood, samples from an administration study using an adeno‐associated virus serotype 6 expressing green fluorescence protein (AAV6‐GFP) were stored at −20°C for 8 months before analysis. The AAV vector was detected in stored plasma samples, following the same detection profile as the fresh plasma samples. The stored blood showed lower overall DNA detection but followed the same detection profile as the plasma samples. This study provides confidence that re‐analysing plasma samples and/or analysing a frozen ‘B’ sample with different matrix such as whole blood after prolonged storage will still result in the detection of gene doping material. Equine whole blood and plasma samples from an administration of AAV‐GFP viral vector were stored at −20°C for 8 months and analysed with qPCR and massively parallel sequencing. The vector detection in stored plasma samples followed the same detection profile as the fresh plasma samples with minimal degradation. Stored blood samples showed lower overall DNA detection but followed the same detection profile as the plasma samples. This study provides confidence in the detection of gene doping material when re‐analysing samples and/or analysing ‘B’ samples for confirmatory analysis.