Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria
Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria
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改进的 PCR 引物可扩增 NC10 细菌的 16S rRNA 基因
DOI：
10.1007/s00253-016-7477-9
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发表时间：
2016-06-01
影响因子：
5
通讯作者：
Hu, Baolan
中科院分区：
文献类型：
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作者：
He, Zhanfei;Wang, Jiaqi;Hu, Baolan
甲烷厌氧氧化-亚硝酸盐还原耦合反应（AOM-NIR）对于缓解甲烷引起的温室效应具有重要的生态意义。负责这种反应的微生物NC 10细菌已在不同的生态系统中被广泛检测到。然而，在常用的NC 10特异性引物202 F和qP 1F中发现了一些缺陷。针对以往引物的缺陷，对引物进行了重新设计和改进。利用改进的引物建立了一种新的套式PCR方法，用于扩增NC 10细菌的16 SrRNA基因。在新的巢式PCR方法中，qP 1 mF/1492 R和1051 F/qP 2 R引物组分别用于第一轮和第二轮。对PCR产物进行了测序，与以前的引物相比，使用新引物获得了更多的NC 10门的操作分类单位（OTU）。用系列稀释法对新的巢式PCR方法进行了灵敏度测试，其检测限约为103拷贝g-1干sed。对于环境样品，与大约105拷贝g-1干sed相比。通过以前的方法。最后，将改进后的引物qP 1 mF用于NC 10细菌丰度的定量PCR（qPCR）检测，结果与同位素示踪法测定的AOM-NIR活性一致。改进的引物能够比以前的引物更有效地扩增NC 10 16 S rRNA基因，并且可用于探索不同系统中NC 10门的微生物群落。
Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 103copies g−1dry sed. for the environmental samples compared to approximately 105copies g−1dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems.