...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied and Environmental Microbiology >Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations
【24h】

Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations

机译：基于荧光原位杂交-流式细胞术-细胞分选的分离和富集I型和II型甲烷营养菌种群的方法

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 104 or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.
机译：基于荧光原位杂交流式细胞术（FISH / FC）的方法是使用纯营养的甲烷营养细菌的人工混合物进行了优化。靶向I型（MG84 / 705探针）和II型（MA450探针）甲烷营养菌的16S rRNA的传统寡核苷酸探针用荧光素或Alexa fluor标记并用于FISH，随后进行荧光激活的FC分析和细胞分选（FACS）。该方法导致从人工混合物中有效分离靶细胞（I型或II型甲烷营养生物）。然后将该方法用于检测和富集天然样品华盛顿湖沉积物中的I型和II型甲烷营养菌种群。从沉积物中提取细胞，固定，然后进行FISH / FC / FACS处理。通过对16S rRNA，pmoA（编码颗粒甲烷单加氧酶的亚基）和fae（编码甲醛激活酶）基因的逆转录酶PCR调查分析了所得的亚群。功能基因分析表明I型和II型甲烷营养菌种群的特异性分离。 16S rRNA基因分析显示，使用I型特异性探针分离的I型甲烷营养菌占亚种群的59％，而使用II型特异性探针分离的II型甲烷营养菌占亚种群的47.5％。我们的数据表明，所描述的FISH / FC / FACS协议可以显着丰富来自复杂自然社区的目标微生物种群，并且可以将这些微生物用于基因测试。我们进一步测试了使用已知基因组序列的人工微生物混合物从有限数量的分选细胞中直接进行全基因组扩增（WGA）的可能性。我们证明了有效的WGA可以通过使用16S rRNA特异性FISH / FC / FACS分离的104个或更多个细胞来实现，而更少的细胞导致WGA的特异性降低。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号