Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control.

IF 2.6 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS
Jillian Maniego, Caitlin Harding, Jocelyn Habershon-Butcher, Pamela Hincks, Graham Stewart, Christopher Proudman, Edward Ryder
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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.

利用数字 PCR 和 qPCR 检测马干血点中的转基因,以控制基因兴奋剂。
由于易于采集、运输和储存,干血斑(DBS)为检测基因治疗滥用(又称基因兴奋剂)提供了一种极具吸引力的替代基质。本研究通过评估不同的目标类型、DNA 提取试剂盒、打孔次数和血管防腐剂,对干血斑中 DNA 的回收率、提取效率和由此产生的检测能力进行了评估。本研究没有评估 DBS 中低拷贝数转基因目标的长期储存稳定性,但值得进一步研究。DNA 定量采用两种检测方法:qPCR 和数字 PCR(dPCR)。与其他提取方法相比,使用 Qiagen Investigator 套件进行六次打孔可获得最佳的总体 DNA 产率。不过,使用三个冲头的DNA提取效率更高。使用 qPCR 和 dPCR 可以检测到添加了 5000 个拷贝/毫升血液的 DBS 中的参考物质(每 3 毫米冲孔中约有 15 个拷贝)。最佳 DNA 提取方案用于定制重组腺相关病毒给药研究的 DBS 样品,结果显示 DBS 样品中成功检测到了载体靶标。
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来源期刊
Drug Testing and Analysis
Drug Testing and Analysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL
CiteScore
5.90
自引率
24.10%
发文量
191
审稿时长
2.3 months
期刊介绍: As the incidence of drugs escalates in 21st century living, their detection and analysis have become increasingly important. Sport, the workplace, crime investigation, homeland security, the pharmaceutical industry and the environment are just some of the high profile arenas in which analytical testing has provided an important investigative tool for uncovering the presence of extraneous substances. In addition to the usual publishing fare of primary research articles, case reports and letters, Drug Testing and Analysis offers a unique combination of; ‘How to’ material such as ‘Tutorials’ and ‘Reviews’, Speculative pieces (‘Commentaries’ and ‘Perspectives'', providing a broader scientific and social context to the aspects of analytical testing), ‘Annual banned substance reviews’ (delivering a critical evaluation of the methods used in the characterization of established and newly outlawed compounds). Rather than focus on the application of a single technique, Drug Testing and Analysis employs a unique multidisciplinary approach to the field of controversial compound determination. Papers discussing chromatography, mass spectrometry, immunological approaches, 1D/2D gel electrophoresis, to name just a few select methods, are welcomed where their application is related to any of the six key topics listed below.
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