肠道菌群核酸提取自动化流程的优化

发布时间：2018-05-13 06:56

本文选题：肠道菌群 + 鸟枪法测序　；参考：《中国生物工程杂志》2017年11期

【摘要】：目的:优化肠道菌群核酸提取自动化流程。方法:收集粪便样本,分别采用手工方法、核酸提取工作站提取核酸,然后利用液体工作站制备PCR反应液进行PCR扩增,最后采用文库工作站建库测序。结果:400μl样品用QIAamp~汶 Fast DNA Stool Mini Kit试剂盒裂解液裂解后,用MagMAX~(TM) Express 96机器提取核酸的方法与用QIAamp~汶 Fast DNA Stool Mini Kit试剂盒手工提取核酸的方法相比,测序得到的序列数基本一致,分析结果也没有明显区别;而单独采用MagMAX~(TM)Viral RNA Isolation Kit试剂盒提取核酸由于样品投入体积受限(50μl)、核酸浓度低、测序得到的序列数太少,不能满足后续的分析要求。结论:通过结合使用两种不同的试剂盒,可以实现核酸提取、PCR反应液配制及文库制备的全流程自动化操作,从而大大提高工作效率和结果的稳定性。
[Abstract]:Objective: to optimize the automated extraction process of intestinal bacterial nucleic acid. Methods: the fecal samples were collected, the nucleic acid was extracted by manual method, and the PCR reaction solution was prepared by liquid workstation for PCR amplification. Finally, the library was constructed and sequenced by library workstation. Results the nucleic acid was extracted by using MagMAXT Express 96 machine after the cleavage of 1: 400 渭 l sample with QIAamp-Wen Fast DNA Stool Mini Kit kit. Compared with the method of manual extraction of nucleic acid with QIAamp-Fast DNA Stool Mini Kit kit, the sequence number obtained by sequencing was basically the same. There was no significant difference in the analysis results, but the MagMAX~(TM)Viral RNA Isolation Kit kit alone was used to extract nucleic acid because of the limited input volume of 50 渭 l of nucleic acid, the low concentration of nucleic acid, and the small number of sequencing sequences, which could not meet the requirements of subsequent analysis. Conclusion: by combining two different kits, the whole process of preparation of nucleic acid extraction PCR reaction solution and the preparation of library can be realized, thus greatly improving the working efficiency and stability of the results.
【作者单位】：安徽医科大学;中国人民解放军军事科学院微生物与流行病研究所;
【基金】：病原微生物生物安全国家重点实验室课题资助项目(SKLPBS1518)
【分类号】：R37

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