Abstract
The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 104 to 2.30 × 105 copies μg−1 DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.
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Acknowledgments
The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (51178444), the National Hi-Technology Development 863 Program of China (2011AA060901), the Hundred-Talent Program of the Chinese Academy of Science (CAS), the Collaborative Innovation Center of Suzhou Nano Science and Technology, the Program for Changjiang Scholars and Innovative Research Team in University, the Fundamental Research Funds for the Central Universities (wk2060190040), and the Research Grants Council of Hong Kong Special Administrative Region, China (project no. CityU 160110).
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New primers design for DAMO archaea. (DOCX 3.95 mb)
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Ding, J., Ding, ZW., Fu, L. et al. New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches. Appl Microbiol Biotechnol 99, 9805–9812 (2015). https://doi.org/10.1007/s00253-015-6893-6
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DOI: https://doi.org/10.1007/s00253-015-6893-6