caa a22 4500 60550086X CHVBK 20210128100558.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6893-6 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6893-6 New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches [Elektronische Daten] [Jing Ding, Zhao-Wei Ding, Liang Fu, Yong-Ze Lu, Shuk Cheng, Raymond Zeng] 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 2.30×105copiesμg−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. Springer-Verlag Berlin Heidelberg, 2015 DAMO nationallicence ANME-2d nationallicence New primer design nationallicence Real-time PCR nationallicence Phylogenetic analysis nationallicence Environmental samples nationallicence Ding Jing Advanced Laboratory for Environmental Research and Technology, USTC-CityU, 215123, Suzhou, China aut Ding Zhao-Wei CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China aut Fu Liang CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China aut Lu Yong-Ze CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China aut Cheng Shuk Advanced Laboratory for Environmental Research and Technology, USTC-CityU, 215123, Suzhou, China aut Zeng Raymond Advanced Laboratory for Environmental Research and Technology, USTC-CityU, 215123, Suzhou, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/22(2015-11-01), 9805-9812 0175-7598 99:22<9805 2015 99 253 https://doi.org/10.1007/s00253-015-6893-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6893-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ding Jing Advanced Laboratory for Environmental Research and Technology, USTC-CityU, 215123, Suzhou, China aut NATIONALLICENCE 700 1- Ding Zhao-Wei CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China aut NATIONALLICENCE 700 1- Fu Liang CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China aut NATIONALLICENCE 700 1- Lu Yong-Ze CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China aut NATIONALLICENCE 700 1- Cheng Shuk Advanced Laboratory for Environmental Research and Technology, USTC-CityU, 215123, Suzhou, China aut NATIONALLICENCE 700 1- Zeng Raymond Advanced Laboratory for Environmental Research and Technology, USTC-CityU, 215123, Suzhou, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/22(2015-11-01), 9805-9812 0175-7598 99:22<9805 2015 99 253