用于粪便DNA检测的样本保存液制作和效果评估

doi:10.3969/j.issn.1004-616x.2021.04.013

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

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. 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. 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/Abstract

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ΔC_t≤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ΔC_t<1；与RNAlater相比，自制保存液保存粪便样本的条带更单一，ΔC_t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 ΔC_t value were less than or equal to 0.2. After freezings and thawings,the agarose gel electrophoresis results remained the same and ΔC_t 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 ΔC_t 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

中图分类号:

R-331

李博, 李改瑞, 赵伟, 彭晓琳, 赵丹, 彭绩. 用于粪便DNA检测的样本保存液制作和效果评估[J]. 癌变·畸变·突变, 2021, 33(4): 312-316.

LI Bo, LI Gairui, ZHAO Wei, PENG Xiaolin, ZHAO Dan, PENG Ji. Evaluation of sample preservation solution for fecal DNA detection[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(4): 312-316.

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用于粪便DNA检测的样本保存液制作和效果评估

Evaluation of sample preservation solution for fecal DNA detection

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