| 王韵淞,陈 杉,沈燚昀,刘琳琳,邹秉杰,宋沁馨,周国华.符合大肠癌早期无创分子诊断要求的粪便DNA样本贮存条件考察[J].现代生物医学进展英文版,2018,(5):852-857. |
| 符合大肠癌早期无创分子诊断要求的粪便DNA样本贮存条件考察 |
| A Study on the Storage Conditions of Stool DNA Samples in the Early Stage of Non-invasive Molecular Diagnosis of Colorectal Cancer |
| Received:May 24, 2017 Revised:June 17, 2017 |
| DOI：10.13241/j.cnki.pmb.2018.05.011 |
| 中文关键词: 大肠癌 无创分子诊断 粪便DNA 保存条件 |
| 英文关键词: Colorectal cancer Noninvasive molecular diagnostics Stool DNA Storage condition |
| 基金项目:国家自然科学基金面上项目（81673390）；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（DQCP2015MS02）；中国药科大学药学基地科研训练及科研能力提高项目（J1310032）；江苏省青蓝工程资助 |
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| 中文摘要: |
| 摘要 目的：考察在不同温度下储存时间和反复冻融对粪便中人基因组DNA含量的影响。方法：1.将粪便样本在室温、4℃、-40℃和-70℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后，使用QIAamp DNA Stool Mini Kit试剂盒提取得到粪便DNA，通过实时荧光定量PCR体系对人KRAS基因定量确定人基因组DNA含量，评价粪便样本不同冻存条件对其中人基因组DNA含量的影响。2.将粪便DNA样本在4℃和-40℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后，评价粪便DNA样本不同冻存条件下对其中人基因组DNA含量的影响。结果：1).粪便样本常温放置2小时，其中人基因组DNA即发生明显降解(P<0.01)，4℃可保存3天左右，-40℃可保存4周，-70℃可保存3个月以上，粪便反复冻融第3次，其中人基因组DNA降解具有统计学意义(P<0.05)。2).粪便DNA 4℃可保存3天，-40℃可保存4周，粪便DNA反复冻融第4次，其中人基因组DNA降解具有统计学意义(P<0.05)。结论：符合大肠癌早期无创分子诊断要求的粪便DNA贮存条件：粪便样本室温收集后尽快保存；短期可处理的粪便样本存放在4℃条件下（3天内）；暂无法处理则存于-40℃（1个月内）；粪便样本长期保存在 -70℃条件下，可保存3个月。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effects of storage time at different temperatures and repeated freezing and thawing on the content of human genomic DNA in stool. Methods: 1. Fecal samples were stored at room temperature, 4℃, -40℃ and -70℃ for different times and stored at -40℃ repeatedly freeze-thawed. Then the fecal DNA was extracted using the QIAamp DNA Stool Mini Kit. Quantita- tive determination of human genomic DNA in human KRAS gene by fluorescence quantitative PCR system was conducted to evaluate the effect of different cryopreservation conditions on human genomic DNA content. 2. The effects of fecal DNA samples on the content of human genomic DNA under different cryopreservation conditions were evaluated after the fecal DNA samples were stored at 4℃ and -40℃ for different times and at -40℃ freeze-thawed. Results: 1. The stool samples stand at room temperature for 2 hours, the human ge- nomic DNA occurred significantly degraded (P<0.01). The stool samples can be stored at 4℃ for 3 days or so, at -40℃ for 4 weeks, at -70℃ for more than 3 months. When the stool samples thawing was repeated for the third time, the human genomic DNA occurred sig- nificantly degradated (P<0.05). 2. Fecal DNA can be stored at 4℃ for 3 days, at -40℃ for 4 weeks, and fecal DNA was frozen and frozen for the fourth time, human genomic DNA occurred significantly degradated (P<0.05). Conclusion: The storage conditions of stool DNA samples in the early stage of non-invasive molecular diagnosis of colorectal cancer. Stool samples are stored as soon as possible after col- lection at room temperature. Short-term disposable stool samples are stored at 4℃ (within 3 days); temporary disposal is stored at -40 ℃(within 1 month). Stool samples are stored for a long period of time at -70℃ for 3 months. |
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