癌变·畸变·突变 ›› 2021, Vol. 33 ›› Issue (4): 312-316.doi: 10.3969/j.issn.1004-616x.2021.04.013

• 技术与方法 • 上一篇    下一篇

用于粪便DNA检测的样本保存液制作和效果评估

李博1, 李改瑞1, 赵伟2, 彭晓琳1, 赵丹1, 彭绩3   

  1. 1. 深圳市南山区慢性病防治院肿瘤伤害与营养科, 广东 深圳 518054;
    2. 海南省疾病预防控制中心, 海南 海口 570203;
    3. 深圳市慢性病防治中心肿瘤防治科, 广东 深圳 518020
  • 收稿日期:2021-04-22 修回日期:2021-06-17 出版日期:2021-07-30 发布日期:2021-07-29
  • 通讯作者: 彭晓琳,E-mail:25731738@qq.com E-mail:25731738@qq.com
  • 作者简介:李博,E-mail:54222538@qq.com。
  • 基金资助:
    深圳市卫计委资助项目(SZGW2017011);南山区卫生科技计划项目(2017007,2019054)

Evaluation of sample preservation solution for fecal DNA detection

LI Bo1, LI Gairui1, ZHAO Wei2, PENG Xiaolin1, ZHAO Dan1, PENG Ji3   

  1. 1. Department of Tumor, Injury and Nutrition, Shenzhen Nanshan Center for Chronic Disease Control, Shenzhen 518054;
    2. Hainan Center for Disease Control and Prevention, 570203;
    3. Department of Cancer Prevention and Treatment, Shenzhen Center for Chronic Disease Control, Shenzhen 518020, Guangdong, China
  • Received:2021-04-22 Revised:2021-06-17 Online:2021-07-30 Published:2021-07-29

摘要: 目的:研发一种粪便样本保存液,可用于结直肠癌早筛人群粪便样本的常温保存与运输,且保存样本可用于后续粪便样本DNA检测。方法:改良配方,配制自制粪便样本保存液,将其用于常温保存粪便样本3和7 d,以及7 d内-80℃冻融一次后,采用琼脂糖凝胶电泳定性及实时荧光定量PCR(qPCR)检测人源看家基因β-globin的DNA拷贝数测试其阻遏DNA降解及微生物增殖的效能,并与RNAlater进行比较。自制粪便样本保存液应用于501例结直肠癌筛查人群,采用qPCR检测人源看家基因ATCB的DNA拷贝数,评估其对粪便DNA的保存效能。结果:自制保存液常温保存粪便样本3和7 d后琼脂糖凝胶电泳结果显示β-globin条带无明显变化,qPCR结果显示ΔCt≤0.2;采用自制保存液保存粪便样本并在7 d内-80℃冻融一次条件下,琼脂糖凝胶电泳结果显示β-globin条带无明显变化,qPCR结果显示ΔCt<1;与RNAlater相比,自制保存液保存粪便样本的条带更单一,ΔCt值变化更小。应用于结直肠癌筛查人群时,自制粪便样本保存液保存的样本合格率>70%,明显优于未使用保存液的样本合格率(<40%)。结论:本研究开发了一种经济有效的粪便样本保存液,既不需要冷链运输,又能较长时间保存粪便DNA完整性并抑制微生物的增殖,为将其用于大样本量结直肠癌人群的早期无创筛查提供研究基础。

关键词: 粪便, 样本保存液, 粪便DNA, 结直肠癌, 早期筛查

Abstract: OBJECTIVE: To develope a solution which could be used for preserving DNA samples in fecal samples from individuals who participated in early colorectal cancer screening. METHODS: An original formula was modified for this investigation. Fecal samples were stored at room temperature for 3 and 7 days,and freezing and thawing at -80℃ within the 7 days. Then,agarose gel electrophoresis (the qualitative method) and real-time fluorescent quantitative PCR (qPCR,the semi-quantitative method) were used for detecting presence of the human housekeeping β-globin gene. Usefulness of our modified preservation solution wascompared to similar international preservation solutions using 501 of the fecal samples. RESULTS: The agarose gel electrophoresis band did not change significantly after the fecal samples were stored at room temperature for 3 and 7 days. The qPCR results as measured using the ΔCt value were less than or equal to 0.2. After freezings and thawings,the agarose gel electrophoresis results remained the same and ΔCt value for the qPCR results was less than 1. Compared with the international preservation solutions,our solution showed more clear bands and less changes of the ΔCt value. Using our solution,effectiveness in preserving fecal samples for DNA analysis was >70% compared to <40% if no preservation solutions were used. CONCLUSION: Our data indicate that our preservation solution was both economical and effective in preserving fecal samples after cold-chain transportation and in inhibiting proliferation of microorganisms. Our solution is therefore useful for use in large-scale colorectal cancer screening.

Key words: feces, sample preservation solution, fecal DNA, colorectal cancer, early screening

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