李鹏;毕学军;汝少国;

• 1:中国海洋大学海洋生命学院
• 2:中国海洋大学海洋生命学院
• 3:青岛理工大学环境与市政工程学院 山东青岛266033
• 4:中国海洋大学海洋生命学院

摘要(Abstract)：

评价超声波法、2种基于SDS的裂解法、改进的化学裂解法、微波法和冻融+玻璃珠+溶菌酶+SDS等6种DNA提取方法对活性污泥微生物多样性PCR-DGGE检测的影响。结果表明,2种不同的DNA纯化方式(溶液和胶纯化)及PCR扩增方式(直接和巢式PCR)对DGGE结果没有明显的影响,但不同的DNA提取方法对DGGE结果有明显的影响,其中2种基于SDS裂解法的DGGE条带较多,其提取的DNA产量(14.85μg/g湿重)和纯度(OD260∶OD280>1.80)也最高;改进的化学裂解法和微波法次之;超声波法、冻融+玻璃珠+溶菌酶+SDS法较少。研究表明,DNA提取方法是影响活性污泥微生物多样性PCR-DGGE检测结果的关键因素,基于SDS的裂解法是用于活性污泥微生物多样性PCR-DGGE研究的一种简便、可靠的总DNA提取方法。

关键词(KeyWords)： 微生物生态学;活性污泥;DNA提取;PCR-DGGE;微生物多样性

Abstract:

Keywords:

基金项目(Foundation): 山东省优秀中青年科学家科研奖励基金项目(2006BSB08027);; 青岛市自然科学基金项目(05-2-JC-57)

作者(Authors): 李鹏;毕学军;汝少国;

参考文献(References)：

• [1]WEN-TSOL,ON-CHIMC,FANG H.Microbial community dynamics duringstart-up of acidogenic anaerobic reactors[J].Wat Res,2002,36(13):3203-3210.
• [2]ZHANG T,FANG H.Phylogenetic diversity of a SRB-rich marine biofilm[J].Appl Microbiol Biotechnol,2001,57(3):437-440.
• [3]LIESACK W,WEYLAND H,STACKEBRANDT E.Potential risks of gene amplification by PCR as determined by16S rDNA analysis of a mixed-culture of strict barophilic bacteria[J].Microbial Ecology,1991,21(1):191-198.
• [4]PICARD C,PONSONNETC,PAGETE,et al.Detection and enumer-ation of bacteria in soil by direct DNAextraction and polymerase chain reaction[J].Appl Environ Microbiol,1992,58(9):2717-2722.
• [5]STACHJEM,BATHES,CLAPPJ P,et al.PCR(SSCPcomparison of16SrDNAsequence diversityin soil DNAobtained using different isola-tion and purification methods[J].FEMS Microbiol Ecol,2001,36(2/3):139-151.
• [6]MURIEL B,WAFAA,VINCENTU,et al.DNAextractionfromacti-vated sludges[J].Current Microbiol,1999,38(6):315-319.
• [7]GAOPingping(高平平),ZHAO Liping(赵立平).DNAextract ion from activated sludge for molecular community analysis[J].Acta Ecologica Sinica(生态学报),2002,11(22):2015-2019.
• [8]XIONG Kairong(熊开容),ZHANG Zhiming(张智明),ZHANG Min(张敏).Extraction of DNA from activated sludge[J].Biotechnology(生物技术),2005,15(4):43-46.
• [9]MILLER D N,BRYANTJ E,MADSENEL,et al.Evaluationand op-timization of DNAextractionand purification proceduresfor soil andsed-iment samples[J].Appl Environ Microbiol,1999,65(11):4715-4724.
• [10]ZHOUJ,BRUNS MA,TIEDJEJ M.DNArecoveryfromsoils of di-verse composition[J].Appl Microbiol Biotechnol,1996,62(2):316-322.
• [11]XING Defeng(邢德峰),RENNanqi(任南琪),GONG Manli(宫曼丽).Application of PCR-DGGEto resolve microbial diversity in bio-hydrogen producingreactor[J].Environmental Science(环境科学),2005,2(26):172-176.
• [12]ORSINI M,ROMANO-SPICA V.A microwave-based method for nu-cleic andisolationfromenvironmental samples[J].Letters in Applied Microbiology,2001,33(1):17-20.
• [13]XIAS Q,YANS,FU Y G,et al.DGGE analysis of16S rDNAof ammonia-oxidizing bacteria in chemical-biological flocculation and chemical coagulation systems[J].Appl Microbiol Biotechnol,2005,69(1):99-105.
• [14]MUYER G,WAAL E Cde,ULTTERLINDENAG.Profiling of com-plex microbial population by denaturing gradient gel electrophoreses analysis of polymerse chain reaction-amplified genes coding for16S rRNA[J].Appl Environ Microbiol,1993,59(3):695-700.
• [15]EICHNER C A,ERB R W,TIMMIS KN,et al.Thermal gradient gel electrophoresis analysis of bio-protection from pollutant shocks in the activated sludge microbial community[J].Appl Environ Microbiol,1999,65(1):102-109.
• [16]BOSSHARD P P,SANTINI Y,GRüTER D,et al.Bacterial diversity and community compositioninthe chemocline of the meromictic alpine Lake Cadagno as revealed by16SrDNAanalysis[J].FEMS Microbiol Ecol,2000,31(2):173-182.
• [17]JULIA R DE LIPTHAY,CHRISTIANE ENZINGER,KAARE JOHNSEN,et al.Impact of DNAextraction method on bacterial com-munity composition measured by denaturing gradient gel electrophoresis[J].Soil Biology&Biochemistry,2004,36(10):1607-1614.
• [18]OGRAMA,SAYLERGS,BARKEYT,et al.The extractionand pu-rification of microbial DNAfromsediments[J].J Microbial,1987,7(2/3):57-66.
• [19]TSAI YL,PARK MJ,OLSONB H,et al.Rapid method for direct extraction of DNA from soil and sediments[J].Appl Environ Microbiol,1991,57(4):1070-1074.