鹌鹑肠道微生物PCR-DGGE方法的优化

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.011

生物技术通报 ›› 2015, Vol. 31 ›› Issue (1): 73-78.doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.011

鹌鹑肠道微生物PCR-DGGE方法的优化

张振华鲍王波² 余冉² 王长永刘燕

（1.环境保护部南京环境科学研究所，南京 210042；2.东南大学能源与环境学院，南京 210096）

收稿日期:2014-06-09 出版日期:2015-01-09 发布日期:2015-01-10
作者简介:张振华，男，博士，研究方向：微生物学；E-mail：zzh@nies.org
基金资助:
转基因生物新品种培育重大专项（2013ZX08011-003，2014ZX08011-003，2013ZX08012-005，2014ZX08012-005），环保公益行业专项（201309038）

Optimization Method for the Extraction of Total DNA from the Intestinal Microbes in Quails

Zhang Zhenhua, Bao Wangbo², Yu Ran², Wang Changyong, Liu Yan

（1. Nanjing Institute of Environmental Sciences，MEP，Nanjing 210042；2. Department of Energy and Environment，Southeast University，Nanjing 210096）

Received:2014-06-09 Published:2015-01-09 Online:2015-01-10

摘要/Abstract

摘要： 通过对鹌鹑肠道微生物总DNA不同提取方法的效率进行比较，探讨DNA质量以及PCR-DGGE反应条件对研究鹌鹑肠道微生物多样性的影响。本试验利用常规的饱和苯酚/氯仿法和3种试剂盒（QIAGEN试剂盒、上海生工试剂盒、天根试剂盒）方法提取鹌鹑肠道微生物总DNA，分别以细菌V3可变区引物和V6-V8可变区引物以及不同退火温度进行PCR-DGGE分析。QIAGEN试剂盒法提取的肠道微生物总DNA的 A₂₆₀/A₂₈₀值在1.8-1.9之间，浓度约200 ng/μL，DGGE微生物多样性条带均匀度均高于其他3种提取方法。此外，利用不同引物扩增的DGGE图谱发现，以V6-V8可变区引物扩增的DGGE图谱条带均匀，分离充分，较V3可变区更能反映鹌鹑肠道微生物种群的多样性；同时，随PCR退火温度的升高，V6-V8可变区的微生物多样性指数下降，V3可变区的微生物多样性则无明显变化。

关键词: 鹌鹑, 肠道微生物, 总DNA提取, 变性凝胶梯度电泳（DGGE）

Abstract: Comparing the effects of total DNA extracted by different methods from intestinal microbes in quails, the study aimed at investigating the influences of DNA quality and PCR-DGGE conditions on intestinal microbial diversity of quail. In this study, the experiment used four methods to extract the DNA. The first was a conventional method for using saturated phenol/chloroform, other three commercially available DNA extraction kits（QIAGEN, SHENGGONG, TIANGEN）were assessed. The ribosomal gene sequences were amplified with V3 and V6-V8 hypervariable region universal primers at different annealing temperature and subjected to denaturing gradient gel electrophoresis（DGGE）. The QIAGEN DNA Kit generated the greatest bacterial species eveness and DNA quantity and quality in compare with other methods（A₂₆₀/A₂₈₀=1.8, 200 ng/μL）. The resulting DGGE profiles were substantially different in terms of the number and relative intensity of the amplification products. The V6-V8 region produced better DGGE profiles than V3 region. The QIAGEN DNA Kit and V6-V8 hypervariable region universal primers will be used in future studies of intestinal microbial diversity of quail.

Key words: quail, ntestinal microbes, DNA extraction, DGGE

张振华,鲍王波,余冉,王长永,刘燕. 鹌鹑肠道微生物PCR-DGGE方法的优化[J]. 生物技术通报, 2015, 31(1): 73-78.

Zhang Zhenhua, Bao Wangbo, Yu Ran, Wang Changyong, Liu Yan. Optimization Method for the Extraction of Total DNA from the Intestinal Microbes in Quails[J]. Biotechnology Bulletin, 2015, 31(1): 73-78.

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鹌鹑肠道微生物PCR-DGGE方法的优化

Optimization Method for the Extraction of Total DNA from the Intestinal Microbes in Quails

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