| 引用本文: | 李雪莲,齐谢敏,邹秉杰,黄青,宋沁馨,周国华.5种试剂盒对血浆游离DNA提取效果的比较[J].中国现代应用药学,2018,35(8):1119-1123. |
| LI Xuelian,QI Xiemin,ZOU Bingjie,HUANG Qing,SONG Qinxin,ZHOU Guohua.Comparison of Five Kits for Cell-free DNA Extraction[J].Chin J Mod Appl Pharm(中国现代应用药学),2018,35(8):1119-1123. |
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| 5种试剂盒对血浆游离DNA提取效果的比较 |
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李雪莲,齐谢敏,邹秉杰,黄青,宋沁馨,周国华
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药物质量与安全预警教育部重点实验室，中国药科大学,南京总医院药理科,南京总医院药理科,南京总医院药理科,药物质量与安全预警教育部重点实验室，中国药科大学,南京总医院药理科
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| 摘要: |
| 目的 筛选一种操作简单、快捷、成本低、提取效率高、易富集到短片段的血浆游离DNA （cell-free DNA，cfDNA）提取试剂盒。方法 向健康人血浆中投入一定量的6种长度各异的非人源DNA，经5种试剂盒提取后，使用实时荧光定量PCR （Real-time quantitative PCR，qPCR）定量，比较5种试剂盒提取回收率的差异。用上述试剂盒提取乳腺癌患者血浆cfDNA，并对其中6种不同长度的Kras基因片段进行定量，比较5种试剂盒对cfDNA提取效率的差异。结合提取成本、耗时等综合评价，最终筛选出最优试剂盒。结果 试剂盒1对>157 bp的片段回收率最高（75.5%~87.4%），提取总cfDNA含量最高[（154.0±10.1） copies·μL-1血浆）]，每个样本提取成本为274元、耗时73 min，但提取过程需要真空泵。试剂盒2对<157 bp的片段回收率在5个试剂盒中最高（31.8%~33.7%），对<145 bp的cfDNA提取效率最高[（81.7±6.2） copies·μL-1血浆）]，提取总cfDNA含量[（137.1±13.9） copies·μL-1血浆）]仅次于试剂盒1，每个样本提取成本为15.6元、耗时43 min，无需特殊仪器。结论 试剂盒1提取的cfDNA总量最高，但成本较高；对于提取片段分布<150 bp的cfDNA，宜选用试剂盒2，且性价比最高。 |
| 关键词: 试剂盒 血浆游离DNA 实时荧光定量PCR 回收率 提取效率 |
| DOI：10.13748/j.cnki.issn1007-7693.2018.08.001 |
| 分类号:R963 |
| 基金项目:国家自然科学基金项目（81673390，81603219，81603196）；江苏省重点研发计划（社会发展）项目（BE2016745）；江苏省基础研究计划（自然科学基金）项目（BK20151445）；药物质量与安全预警教育部重点实验室资助项目（DQCP2017MS01）；江苏省青蓝工程 |
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| Comparison of Five Kits for Cell-free DNA Extraction |
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Li Xuelian,Qi Xiemin,Zou Bingjie,Huang Qing,Song Qinxin and Zhou Guohua
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Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education,China Pharmaceutical University,Department of pharmacology, Jinling Hospital, Medical School of Nanjing University,Department of pharmacology, Jinling Hospital, Medical School of Nanjing University,Department of pharmacology, Jinling Hospital, Medical School of Nanjing University,Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education,China Pharmaceutical University,Department of pharmacology, Jinling Hospital, Medical School of Nanjing University
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| Abstract: |
| OBJECTIVE To screen a cell-free DNA(cfDNA) extraction kit which is compatible with the requirements of easy-processing, speediness, low price, and high extraction efficiency to enrich short DNA fragments. METHODS In order to compare recovery among five DNA extraction kits, six kinds of non-human derived DNA fragments were added into healthy human plasma. Those DNA fragments were extracted by five DNA extraction kits and then quantified by real-time quantitative PCR(qPCR). Next, plasma cfDNA originated from breast cancer patients were extracted by DNA extraction kits above. After quantification of six kinds of Kras gene fragments, extraction efficiency was estimated. Besides, evaluation related to cost and process time were investigated. In summary, optimal extraction kits were determined. RESULTS Kit 1 showed the highest recovery with fragments >157 bp(75.5%-87.4%), and the highest quantities of total cfDNA[(154.0±10.1)copies·μL-1 plasma)]. The cost of each sample was 274 CNY and it spent 73 min on processing one sample. However, a vacuum pump was required for the protocol. Moreover, Kit 2 not only exhibited the highest recovery of the fragments <157 bp(31.8%-33.7%) among five kits, but also gave the highest quantities of cfDNA <145 bp[(81.7±6.2)copies·μL-1 plasma)]. Total quantities of cfDNA[(137.1±13.9)copies·μL-1 plasma)] were slightly lower than Kit 1. The extraction cost per sample was 15.6 CNY, which took 43 min. Notably, special equipment was unnecessary. CONCLUSION Despite the fact Kit 1 is expensive, it can achieve the highest total cfDNA quantities. As for fragments <150 bp, Kit 2 is an excellent and cost-effective choice. |
| Key words: kit cell-free DNA real-time quantitative PCR recovery extraction efficiency |
