Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification
Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification
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DOI：
10.1016/j.mimet.2019.03.020
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发表时间：
2019-05-01
影响因子：
2.2
通讯作者：
Chee-Sanford, Joanne
中科院分区：
文献类型：
--
作者：
Cannon, Jordan;Sanford, Robert A.;Chee-Sanford, Joanne
异化硝酸盐还原为铵（DNRA）是目前已知的陆地生态系统中比以前认为的更普遍的过程。关键酶，五血红素细胞色素c亚硝酸盐还原酶NrfA与呼吸亚硝酸盐氨化，编码的nrfA基因在广泛的细菌的共生性。缺乏可靠和全面的分子工具来检测环境样品中的不同nrfA，阻碍了有意义地表征环境系统中DNRA遗传潜力的努力。在这项研究中，进行了修改，以优化先前设计的PCR引物的扩增效率，靶向保守的第三和第四血红素结合结构域之间的NrfA的诊断区域，并增加覆盖范围，包括检测环境相关的Geobacteraceae样nrfA。使用引物与隶属于18个不同进化枝的> 270个细菌nrfA基因的比对，对引物序列的修饰提高了覆盖率，最小化了扩增假象，并从参考、土壤和地下水DNA中产生了预测的产物大小。扩增子的Illumina测序显示，基于翻译序列的比对，从环境DNA中成功回收nrfA基因片段。本研究开发的新引物在PCR反应中更有效，尽管具有高GC含量的基因靶影响效率。此外，引物具有更宽的检测谱，并经过严格验证，可用于检测自然环境中的nrfA。这些适用于常规PCR、qPCR和PCR访问阵列技术，这些技术允许下游高通量测序平台进行多重基因扩增。
Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to > 270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms.