Lin Chenshuo, Xu Hang, Qin Wei, Xu Shaoyi, Tang Xiufeng, Kuang Lu, Wang Xinxin, Jiang Bin, Chen Junhui, Shan Jun, Adams Jonathan, Qin Hua, Wang Baozhan
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, China.
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lake of Ministry of Education, College of Environment, Hohai University, Nanjing, China.
Front Microbiol. 2020 Sep 30;11:560942. doi: 10.3389/fmicb.2020.560942. eCollection 2020.
After the discovery of complete ammonia-oxidizing (comammox) , detection and assessments of the contribution of comammox communities to nitrogen cycling are in great demand. PCR-based approach, a common method for the detection of comammox, depends strongly on accurate amplification of the genes from the original DNA samples using appropriate primers. In this study, we reported an evaluation of the performance of two commonly used primer sets, Ntsp-amoA 162F/359R and comaA/B-244f/659r, for amplifying the comammox genes from three representative wetland soils in China [Sangsang (SS), Sanjiang (SJ), and Xianghai (XH)]. Our results demonstrated the two primer sets could both successfully amplify the clades with high relative abundances (RA), and further revealed a broadly similar diversity and community composition of dominant comammox operational taxonomic units (OTUs) (RA ≥ 1%) in each of the three wetland soils. However, the clades with low RA, such as the clade A (1.26%) in SJ and the clade B (11.54%) in XH that were recovered by metagenomics analysis, failed to be amplified using comaA/B-244f/659r, but were successfully amplified and sequenced using Ntsp-amoA 162F/359R. It indicated that, compared to comaA/B-244f/659r, Ntsp-amoA 162F/359R was more sensitive to the clades with low RA. However, it is worth noting that Ntsp-amoA 162F/359R would overestimate the RA of some rare clades. For example, the RAs of clade B in XH were overestimated by 32-fold. Furthermore, high levels of non-target amplification were detected via gel electrophoresis using both primer sets, especially for comammox Clade B genes, implying that we should treat qPCR results based on these primers with caution. Taken together, our study comprehensively compared the performance of the two primer sets on the sensitivity and specificity of amplifying comammox genes in three wetland soils, pointing out the necessity of further development of new primers for the efficient and accurate detection of comammox in various environments.
在发现完全氨氧化菌（comammox）后，对comammox群落对氮循环贡献的检测和评估需求巨大。基于PCR的方法是检测comammox的常用方法，它强烈依赖于使用合适的引物从原始DNA样本中准确扩增基因。在本研究中，我们报告了对两种常用引物组Ntsp-amoA 162F/359R和comaA/B-244f/659r从中国三种代表性湿地土壤[桑桑（SS）、三江（SJ）和向海（XH）]中扩增comammox基因性能的评估。我们的结果表明，这两种引物组都能成功扩增出相对丰度较高的进化枝，并且进一步揭示了三种湿地土壤中优势comammox可操作分类单元（OTU）（相对丰度≥1%）的多样性和群落组成大致相似。然而，宏基因组分析检测到的相对丰度较低的进化枝（如SJ中的进化枝A，相对丰度为1.26%；XH中的进化枝B，相对丰度为11.54%），使用comaA/B-244f/659r未能扩增出来，但使用Ntsp-amoA 162F/359R成功扩增并测序。这表明，与comaA/B-244f/659r相比，Ntsp-amoA 162F/359R对相对丰度较低的进化枝更敏感。然而，值得注意的是，Ntsp-amoA 162F/359R会高估一些稀有进化枝的相对丰度。例如，XH中进化枝B的相对丰度被高估了32倍。此外，使用这两种引物组通过凝胶电泳都检测到了高水平的非靶标扩增，尤其是对于comammox进化枝B基因，这意味着我们应该谨慎对待基于这些引物的qPCR结果。综上所述，我们的研究全面比较了这两种引物组在三种湿地土壤中扩增comammox基因的敏感性和特异性，指出有必要进一步开发新的引物，以高效、准确地检测各种环境中的comammox。
