title: New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches.
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 × 10(4) to 2.30 × 10(5) 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.
authors: Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, 215123, People's Republic of China.
authors: Jing
authors: J
authors: Ding
authors: CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, People's Republic of China.
authors: Zhao-Wei
authors: ZW
authors: Ding
authors: CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, People's Republic of China.
authors: Liang
authors: L
authors: Fu
authors: CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, People's Republic of China.
authors: Yong-Ze
authors: YZ
authors: Lu
authors: Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, 215123, People's Republic of China.|State Key Laboratory in Marine Pollution, Department of Biomedical Science, City University of Hong Kong, Kowloon, Hong Kong.
authors: Shuk H
authors: SH
authors: Cheng
authors: Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, 215123, People's Republic of China. rzeng@ustc.edu.cn.|CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, People's Republic of China. rzeng@ustc.edu.cn.
authors: Raymond J
authors: RJ
authors: Zeng
mesh_terms: D000693:Anaerobiosis; D001105:Archaea / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics*; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D008697:Methane / Q000378:metabolism*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D008697:Methane
keywords: ANME-2d; DAMO; Environmental samples; New primer design; Phylogenetic analysis; Real-time PCR