A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses.,Drug Testing and Analysis

当前位置： X-MOL 学术 › Drug Test. Anal. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses.
Drug Testing and Analysis ( IF 2.6 ) Pub Date : 2020-08-06 , DOI: 10.1002/dta.2907
Hiu Wing Cheung ₁ , Kin-Sing Wong ₁ , Venus Y C Lin ₁ , Terence S M Wan ₁ , Emmie N M Ho ₁

Affiliation

The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non‐human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false‐negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO‐plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO‐plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.

中文翻译：

人促红细胞生成素 (EPO) 转基因的双重 qPCR 检测，用于控制马的基因掺杂。

滥用基因操作技术来提高运动成绩被称为基因兴奋剂，这在人类运动、赛马和马术运动中是被禁止的。尽管已经开发了许多 qPCR 检测，但大多数检测使用来自人类、非人类灵长类动物和小鼠的基因组 DNA (gDNA) 作为背景，它们可能不适用于测试马样本。本研究旨在开发一种 qPCR 检测方法，用于检测马血细胞中的人促红细胞生成素 (hEPO) 转基因，其中基因治疗中使用的病毒载体可以存在数月。对于hEPO转基因的检测，比较了hEPO的三组引物和水解探针的性能。一组在马 gDNA 存在下显示出足够的特异性、灵敏度、扩增效率和动态检测范围。该测定与检测马微管蛋白 α 4A (TUBA4A) 基因作为内源性内部对照进行双重检测，以防止由于提取的 DNA 和/或 qPCR 实验变异的回收和储存不佳而导致假阴性结果。对于 hEPO 质粒的提取，QIAGEN Gentra Puregene 血液试剂盒显示可以从加标的马血细胞中回收大部分 (62%) 的 hEPO 质粒。双重 qPCR 检测的特异性和检测限 (LOD) 根据 MIQE 指南确定。这些发现支持将这种双链 qPCR 检测应用于检测马血细胞中的 hEPO 转基因。对于 hEPO 质粒的提取，QIAGEN Gentra Puregene 血液试剂盒显示可以从加标的马血细胞中回收大部分 (62%) 的 hEPO 质粒。双重 qPCR 检测的特异性和检测限 (LOD) 根据 MIQE 指南确定。这些发现支持将这种双链 qPCR 检测应用于检测马血细胞中的 hEPO 转基因。对于 hEPO 质粒的提取，QIAGEN Gentra Puregene 血液试剂盒显示可以从加标的马血细胞中回收大部分 (62%) 的 hEPO 质粒。双重 qPCR 检测的特异性和检测限 (LOD) 根据 MIQE 指南确定。这些发现支持将这种双链 qPCR 检测应用于检测马血细胞中的 hEPO 转基因。

更新日期：2020-08-06

点击分享查看原文

点击收藏

阅读更多本刊最新论文本刊介绍/投稿指南11