School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Biochem Soc Trans. 2011 Jan;39(1):321-6. doi: 10.1042/BST0390321.
Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.
通用引物可用于检测生物氮循环中几乎每一个反应所需的细菌基因，唯一明显的例外是 napA（周质硝酸盐还原酶的钼蛋白编码基因），它编码周质硝酸盐还原酶。我们采用迭代方法，首次成功设计了三个正向寡核苷酸引物和一个反向引物，这三个引物可在三个独立的 PCR 中从所有 5 组变形菌纲扩增 napA DNA。目前在 NCBI（美国国立生物技术信息中心）数据库中列出的所有 140 个 napA 序列都预计可被这些引物对中的一个或多个扩增。我们证明，这两对引物也可从人粪便中分离的 DNA 扩增出预测大小的 PCR 产物，这证实了它们能够从混合群体中定向扩增 napA 片段的能力。通过高通量测序对所得扩增子进行分析，将能够很好地估计任何群落中硝酸盐还原菌的范围和相对丰度，这仅受对高度多样化的目标进行分子特征描述的 PCR 方法固有的任何不可避免的偏差的影响。
