| 王决恒,周宇荀,李 凯,肖君华.游离线粒体拷贝数检测方法建立及应用[J].现代生物医学进展英文版,2022,(14):2607-2615. |
| 游离线粒体拷贝数检测方法建立及应用 |
| Establishment and Application of Detection Method for Cell Free Mitochondrial Copy Number |
| Received:January 21, 2022 Revised:February 17, 2022 |
| DOI：10.13241/j.cnki.pmb.2022.14.002 |
| 中文关键词: 线粒体DNA 荧光定量PCR TaqMan探针 外周血游离DNA |
| 英文关键词: Mitochondrial DNA Real-time quantitative PCR TaqMan Probe Circulating cell free DNA |
| 基金项目:国家自然科学基金项目（31772550）；上海市科委基金资助项目（17140903102） |
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| 中文摘要: |
| 摘要 目的：线粒体在生理和病理过程中都起着重要作用，线粒体破碎后形成的游离线粒体与一系列疾病密切相关。然而，人体内游离线粒体的含量较低很难被稳定抽提且易降解等因素导致游离线粒体拷贝数检测具有极大挑战。本研究拟建立一种快速、准确检测外周血游离线粒体拷贝数定量PCR技术。方法：通过多重荧光定量PCR技术在SLAN?-96S全自动医用PCR分析系统上检测人外周血游离线粒体拷贝数，构建新的游离线粒体检测方案。游离核基因在人体外周血中的稳定性远大于游离线粒体，因此使用多拷贝参考基因YH-1（300拷贝）检测游离核基因作为对照组。结果：成功建立了核基因标准曲线和线粒体标准曲线，并筛选游离线粒体拷贝数检测最佳引物扩增片段长度为82bp、血清有效分离时间在2h内、血清最佳分离方案为1600 r/min 离心10 min再16000 r/min 离心10 min、磁珠法游离核酸抽提试剂盒抽提游离核酸得率最高的新流程。利用新方案对100 例不同年龄段的随机人群外周血抽提游离线粒体拷贝数进行检测，结果显示30-79岁游离线粒体拷贝数与年龄之间的相关性参数为｜R｜= 0.18、P value = 0.077，游离线粒体拷贝数与性别之间的相关性参数为｜R｜= 0.27、P value = 0.061即游离线粒体拷贝数与年龄和性别均无显著相关性，研究结果与报道一致。结论：表明优化后的方案可稳定检测游离线粒体拷贝数，提供了一种快速、准确检测游离线粒体拷贝数的方法。 |
| 英文摘要: |
| ABSTRACT Objective: Mitochondria play an important role in physiological and pathological processes. Free mitochondria formed after mitochondrial fragmentation are closely related to a series of diseases. However, the low content of free mitochondria in human body is difficult to be extracted stably and easy to degrade, which leads to great challenges in detecting the copy number of free mitochondria. This study is to establish a rapid and accurate quantitative PCR technique for detecting the copy number of free mitochondria in peripheral blood. Methods: The copy number of free mitochondria in human peripheral blood was detected by multiplex fluorescence quantitative PCR on SLAN ?-96S automatic medical PCR analysis system, and a new detection scheme of free mitochondria was constructed. The stability of free nuclear genes in human peripheral blood is much greater than that of free mitochondria, so the free nuclear genes were detected by multi-copy reference gene YH-1 (300 copies) as control group. Results: Nuclear gene standard curve and mitochondrial standard curve were successfully established, and the best primer amplified fragment length for detecting free mitochondrial copy number was 82bp, the effective separation time of serum was within 2h, the best separation scheme of serum was centrifuged for 10 min at 1600 r/min and then centrifuged for 10 min at 16000 r/min, and the new process of extracting free nucleic acid with magnetic bead method was the highest. The copy number of free mitochondria extracted from peripheral blood of 100 random people of different ages was detected by the new scheme, The results showed that the correlation parameters between free mitochondria copy number and age were R = 0.18, P value = 0.077, and the correlation parameters between free mitochondria copy number and sex were R = 0.27, P value = 0.061, that is, there was no significant correlation between free mitochondria copy number and age and sex. The results were consistent with the reports. Conclusion: The optimized scheme can detect the copy number of free mitochondria stably, and provides a rapid and accurate method for detecting the copy number of free mitochondria. |
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