根管感染中产甲烷古细菌基于mcrA序列的系统发育分析

›› 2014, Vol. 5 ›› Issue (4): 181-184.

根管感染中产甲烷古细菌基于mcrA序列的系统发育分析

韦艳霞¹,郑纪伟²,杨锋³,刘佃滨³

1. 徐州医学院
2. .徐州医学院口腔医学院
3. 徐州医学院口腔医学院

收稿日期:2014-10-29 修回日期:2014-12-09 出版日期:2014-12-25 发布日期:2015-01-06
通讯作者: 韦艳霞 E-mail:weiyx2007@aliyun.com
基金资助:
国家自然科学基金;江苏省自然科学基金;江苏省高校自然科学基金项目

Phylogenetic analysis of Methanogenic archaea based on mcrA sequence data in endodontic infections

Received:2014-10-29 Revised:2014-12-09 Online:2014-12-25 Published:2015-01-06

摘要/Abstract

摘要： 目的：分析根管感染中产甲烷古细菌的多样性，建立基于功能基因——甲基辅酶M还原酶（methyl coenzyme M reductase，MCR）基因α亚基（mcrA）的系统发育树。方法：利用一对特异性引物选择性扩增感染根管中产甲烷古细菌的mcrA片段，在此基础上建立mcrA克隆文库。通过蓝白斑筛选的方法，筛选出阳性重组克隆子，进一步通过基因测序获得重组克隆子中的插入片段mcrA序列。利用“Clustalx”和“Mega 4”软件包分析mcrA序列，对其进行同源性比较和系统发育学分析。结果：4例根管样本的其中1例检出了产甲烷古细菌；构建了基于mcrA序列的系统发育树。随机选出的8个mcrA克隆片段高度同源，均为类口腔甲烷短杆菌序列型。结论：本例根管感染中产甲烷古细菌的多样性局限于类口腔甲烷短杆菌序列型。

Abstract: Objective： To analyze the diversity of Methanogenic archaea in endodontic infections and establish the phylogenetic tree based on functional gene mcrA. Methods：Using PCR and a pair of specific primers，mcrA was selectively amplified from total genomic DNA extracted from the microbial community in the root canal of endodontic infections. Positive clones were selected by blue-white screening, and the mcrA sequences were determined by DNA sequencing. ‘Clustalx’and ‘Mega 4’ software bags were used to analyze mcrA sequences. Phylogenetic analysis was carried out and the phylogenetic tree of methanogenic archaea was established. Results：Four root canals of endodontic infections were analyzed. We found one case positive for Methanogenic archaea. A phylogenetic tree based on functional gene mcrA was established. The mcrA fragments in endodontic infections showed high sequence similarity with Methanobrevibacter oralis. Conclusions： The diversity of Methanogenic archaea in the one sample from endodontic infections in this study was confined to Methanobrevibacter oralis-like sequence types.

韦艳霞郑纪伟杨锋刘佃滨. 根管感染中产甲烷古细菌基于mcrA序列的系统发育分析[J]. , 2014, 5(4): 181-184.

参考文献

[1]Vianna ME, Conrads G, Gomes BP, et al.Identification and quantification of archaea involved in primary endodontic infections[J].J Clin Microbiol,2006,44(4):1274-1282.
[2]Glaser P, Sakamoto H, Bellalou J, et al.Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis[J].EMBO J,1988,7(12):3997-4004.
[3]Vickerman MM1, Brossard KA, Funk DB, et al.Phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections[J].J Med Microbiol,2007,56(Pt 1):110-118.
[4]González R, Klaassens ES, Malinen E, et al.Differential transcriptional response of Bifidobacterium longum to human milk, formula milk, and galactooligosaccharide[J].Appl Environ Microbiol,2008,74(15):4686-4694.
[5]Scanlan PD, Shanahan F, Marchesi JR.Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis[J].BMC Microbiol,2008,8:79-.
[6]Hales BA, Edwards C, Ritchie DA, et al.Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis[J].Appl Environ Microbiol,1996,62(2):668-675.
[7]Mater DD1, Langella P, Corthier G, et al.A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice[J].J Mol Microbiol Biotechnol,2008,14(1-3):123-127.
[8]Lepp PW, Brinig MM, Ouverney CC, et al.Methanogenic Archaea and human periodontal disease[J].Proc Natl Acad Sci U S A,2004,101(16):6176-6181.
[9]Kulik EM1, Sandmeier H, Hinni K, et al.Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis[J].FEMS Microbiol Lett,2001,196(2):129-133.
[10]Robichaux M, Howell M, Boopathy R.Methanogenic activity in human periodontal pocket[J].Curr Microbiol,2003,46(1):53-58.
[11]Conway de Macario E, Macario AJ.Methanogenic archaea in health and disease: a novel paradigm of microbial pathogenesis[J].Int J Med Microbiol,2009,299(2):99-108.