Chelex 100树脂快速提取动物组织基因组DNA方法的评估

doi:10.11853/j.issn.1003.8280.2019.03.013

中国媒介生物学及控制杂志 ›› 2019, Vol. 30 ›› Issue (3): 286-291.DOI: 10.11853/j.issn.1003.8280.2019.03.013

Chelex 100树脂快速提取动物组织基因组DNA方法的评估

栗冬梅¹, 梁艳林^1,2, 宋秀平¹, 朱彩英¹, 康央^1,3

1 中国疾病预防控制中心传染病预防控制所媒介生物控制室, 传染病预防控制国家重点实验室, 感染性疾病诊治协同创新中心, 北京 102206;
2 山东第一医科大学, 山东泰安 271016;
3 山东大学生命科学学院, 山东济南 250100

收稿日期:2019-02-11 出版日期:2019-06-20 发布日期:2019-06-20
作者简介:栗冬梅,女,研究员,主要从事巴尔通体及相关传染病防治研究,Email:lidongmei@icdc.cn
基金资助:
国家科技重大专项（2017ZX10303404，2018ZX10712001）；传染病预防控制国家重点实验室项目（2018SKLID802）

Evaluation of Chelex 100 resin method for rapid extraction of genomic DNA from animal tissues

LI Dong-mei¹, LIANG Yan-lin^1,2, SONG Xiu-ping¹, ZHU Cai-ying¹, KANG Yang^1,3

1 State Key Laboratory of Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
2 Shandong First Medical University;
3 School of Life Sciences, Shandong University

Received:2019-02-11 Online:2019-06-20 Published:2019-06-20
Supported by:
Supported by the National Science and Technology Major Project of China (No. 2017ZX10303404,2018ZX10712001) and the Project of State Key Laboratory of Infectious Disease Prevention and Control (No. 2018SKLID802)

摘要/Abstract

摘要： 目的应用基于Chelex 100树脂的6种不同处理方法提取动物组织核酸，比较6种处理之间及与磁珠法之间的差异，筛选适用于现场检测、简便快速的动物组织基因组DNA提取方法。方法取适量啮齿动物肝组织，研磨或剪碎后加入5％Chelex 100树脂悬液，应用6种不同的裂解与吸附步骤处理该悬液；经离心或静置处理后收获上清液即为组织基因组DNA，检测其浓度与纯度；以此DNA为模板，应用啮齿动物线粒体细胞色素C氧化酶亚基Ⅰ（COⅠ）基因的引物、巴尔通体通用引物和TaqMan探针扩增COⅠ基因片段和巴尔通体的tmRNA基因片段；比较6种Chelex 100树脂处理方法之间及Chelex 100树脂法与磁珠法提取的基因组DNA浓度、纯度及扩增所得循环值（quantification cycle，C_q）的差异。结果基于Chelex 100树脂的6种不同处理方法（C1～C6）所获DNA模板浓度在203.93～769.86 ng/μl之间，比磁珠法提取的DNA模板浓度偏高；Chelex 100树脂法提取的DNA模板吸光度（A）值A260/A280比值在1.25～1.47之间，磁珠法的A260/A280比值在1.85～1.95之间，C1～C6之间无差别，但纯度低于磁珠法；电泳图显示，相较于磁珠法，Chelex 100树脂法提取的DNA模板没有明确DNA条带，前者DNA条带明显；COⅠ基因的扩增产物电泳带清晰且长度准确；对Chelex 100树脂法的DNA模板进行荧光定量PCR扩增，各样品均可获得正常C_q值，但略高于磁珠法。结论应用Chelex 100树脂法提取动物组织基因组DNA简便、高效，适用于常规PCR及TaqMan探针荧光定量PCR方法鉴定宿主动物及直接检测动物组织DNA中的病原体。

关键词: Chelex 100, 动物组织, 脱氧核糖核酸, 实时荧光定量PCR, 病原体, 即时检验

Abstract: Objective To compare Chelex 100 resin method (six different processing procedures) with the commercial automated magnetic bead-based DNA purification method (the standard method for DNA extraction) for the extraction of genomic DNA from animal tissues, and to identify a simple and rapid method for on-site testing of genomic DNA from animal tissue samples. Methods An appropriate amount of rodent liver tissue was ground or cut prior to adding 5% Chelex 100 resin suspension. The mixture of homogenized tissue with 5% Chelex 100 resin suspension was treated according to six different processing procedures of lysis and adsorption. After centrifugation or static treatment, the concentration and purity of genomic DNA in supernatant were measured. Using the genomic DNA as the templates, the primers of mitochondrial cytochrome c oxidase subunit I (COI) gene for rodents, the universal primers for Bartonella spp. and the TaqMan probe were used to amplify the COI gene fragment and the tmRNA gene fragment of Bartonella spp.. The concentration and purity of genomic DNA and the quantification cycle (C_q) value were compared between the six processing procedures based on Chelex 100 resin as well as between the Chelex 100 resin method and the magnetic bead method. Results The concentration of the DNA templates obtained using the six different processing procedures (C1-C6) based on Chelex 100 resin ranged from 203.93 ng/μl to 769.86 ng/μl, which was higher than that obtained using the magnetic bead method. The A260/A280 ratios of DNA templates extracted using the Chelex 100 resin method and the magnetic bead method ranged from 1.25 to 1.47 and 1.85 to 1.95, respectively. There was no difference in A260/A280 ratio between the C1-C6 based on Chelex 100 resin, but the purity of DNA templates extracted using the Chelex 100 resin method was lower than that of DNA templates extracted using the magnetic bead method. The electrophoretograms showed that the bands of the DNA templates extracted using the magnetic bead method were more distinct than those extracted using the Chelex 100 resin method and there was a clear and accurate band for amplification product of the COⅠ gene. The quantitative real-time PCR amplification results showed that the DNA templates obtained using the Chelex 100 resin method had a normal C_q value, which was slightly higher than that of the DNA templates obtained using the magnetic bead method. Conclusion The Chelex 100 resin method for extracting genomic DNA of animal tissues is simple and efficient, which is suitable for conventional PCR and TaqMan fluorescent probe-based quantitative real-time PCR assay to identify host animal species and directly detect pathogens in animal tissue samples.

Key words: Chelex 100, Animal tissue, Deoxyribonucleic acid, Quantitative real-time PCR, Pathogen, Point-of-care testing

中图分类号:

S443

栗冬梅, 梁艳林, 宋秀平, 朱彩英, 康央. Chelex 100树脂快速提取动物组织基因组DNA方法的评估[J]. 中国媒介生物学及控制杂志, 2019, 30(3): 286-291.

LI Dong-mei, LIANG Yan-lin, SONG Xiu-ping, ZHU Cai-ying, KANG Yang. Evaluation of Chelex 100 resin method for rapid extraction of genomic DNA from animal tissues[J]. Chines Journal of Vector Biology and Control, 2019, 30(3): 286-291.

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Chelex 100树脂快速提取动物组织基因组DNA方法的评估

Evaluation of Chelex 100 resin method for rapid extraction of genomic DNA from animal tissues

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