脱腐预处理对土壤总DNA提取质量的影响  

Effects of pretreatment with humic acid removal on extraction of total soil DNA

作　　者：刘佳[1] 周略[1] 李红萍[1] 程学敏[1] 李庆波[1] 丁记者 朱静媛[1] 

机构地区：[1]郑州大学公共卫生学院环境卫生学教研室,河南郑州450001

出　　处：《环境与健康杂志》2015年第10期910-913,共4页Journal of Environment and Health

基　　金：国家自然科学基金青年基金(81202174)

摘　　要：目的探索一种提高土壤总DNA提取质量的简易脱腐方法。方法于2014年3月,采集河南省某市农田4个采样区的混合土样(07、08、1A、2A)。分别用脱腐预处理后试剂盒提取(预处理组)和直接试剂盒提取法(未预处理组)提取土样总DNA,对保守序列16S r DNA和非保守序列(抗生素抗性基因sulⅡ、tet M、tet C和1类整合子酶基因int I1)进行PCR扩增。采用Gene Genius凝胶成像系统定量分析软件Gene Tools进行灰度值分析。结果预处理组和未预处理组提取的各个土壤总DNA均可扩增出16S r DNA,且PCR产物灰度值差异无统计学意义(均P>0.05);非保守序列sulⅡ、tet C和int I1基因的PCR扩增产物灰度值在两组中差异无统计学意义(均P>0.05);预处理组tet M基因的表达水平高于未预处理组,差异有统计学意义(P<0.05)。未预处理组的sulⅡ、tet M、tet C和int I1基因PCR扩增产物中均检测出了非特异性条带,而预处理组目的条带单一、较少非特异性条带。结论脱腐预处理方法提取的土壤总DNA更有利于后续分子生物学实验。Objective To develop a simple method of pre-removing humic acids for improving the quality of DNA extracted from soil samples. Methods Mixed soil samples（07,08,1A,2A） were collected from four pieces of agricultural soil in certain village of He＇nan province in March, 2014. Total DNA of soil samples were extracted by soil DNA kit with pretreated by humic acid removal agent（pretreated group） or without（non-pretreated group） respectively. Conservative sequence such as 16 S r DNA gene and non conservative sequence（antibiotic resistance genes, such as sulⅡ,tet M,tet C and class 1 integrase gene int I1） were amplified by PCR. Gray levels of PCR products were compared by using the Gene Tools software of Gene Genius gel imaging system. Results 16 S r DNA gene was successfully amplified from both groups and differences of gray levels of PCR products were not significant between two groups（P〉0.05）. Gray levels of PCR products of non conservative sequence gene sulⅡ,tet C and int I1 showed no significant difference between two groups（P〉0.05）. Gray level of tet M was significant higher in pretreated group than that in non-pretreated group（P〈0.05）. Nonspecific PCR amplification strips of sulⅡ,tet M,tet C and Int I1 were found in nonpretreated group while target gene amplification strip was single and there were less nonspecific strips in pretreated group.Conclusion Extraction of total soil DNA with pretreatment by humic acid removal is helpful to subsequent molecular biology experiment.

关 键 词：土壤 总DNA 脱腐预处理 16SrDNA 抗生素抗性基因 

分 类 号：R994.6[医药卫生—毒理学]