Appl Microbiol Biotechnol (2013) 97:10155–10162 DOI 10.1007/s00253-013-5260-8
METHODS AND PROTOCOLS
A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches Ping Han & Ji-Dong Gu
Received: 6 August 2013 / Revised: 10 September 2013 / Accepted: 11 September 2013 / Published online: 17 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract A new pmoA gene-based PCR primer set was designed for detection of nitrite-dependent anaerobic oxidation of methane (n-damo) bacteria from four different ecosystems, namely rice paddy soil, freshwater reservoir, reed bed, and sludge from wastewater treatment plant. This primer set showed high specificity and efficiency in recovering n-damo bacteria from these diverse samples. The obtained sequences showed 88–94 and 90–96 % similarity to nucleotide and amino acid sequences, respectively, with the known NC10 phylum bacterium. According to the UniFrac principal coordinates analysis (PCoA), DNA sequences retrieved by the new PCR primer set in this study formed a separate group from the reported sequences, indicating higher diversity of ndamo in the environment. This newly designed PCR primer is capable of amplifying not only the currently known n-damo bacteria but also those that have not been reported, providing new information on the ecological diversity and distribution of this group of microorganisms in the ecosystem. Keywords Anaerobic methane oxidation . pmoA gene . n-damo . PCR primer
Introduction Anaerobic methane oxidizing coupled to denitrification is possible in theory both thermodynamically and biochemically P. Han : J.
METHODS AND PROTOCOLS
A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches Ping Han & Ji-Dong Gu
Received: 6 August 2013 / Revised: 10 September 2013 / Accepted: 11 September 2013 / Published online: 17 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract A new pmoA gene-based PCR primer set was designed for detection of nitrite-dependent anaerobic oxidation of methane (n-damo) bacteria from four different ecosystems, namely rice paddy soil, freshwater reservoir, reed bed, and sludge from wastewater treatment plant. This primer set showed high specificity and efficiency in recovering n-damo bacteria from these diverse samples. The obtained sequences showed 88–94 and 90–96 % similarity to nucleotide and amino acid sequences, respectively, with the known NC10 phylum bacterium. According to the UniFrac principal coordinates analysis (PCoA), DNA sequences retrieved by the new PCR primer set in this study formed a separate group from the reported sequences, indicating higher diversity of ndamo in the environment. This newly designed PCR primer is capable of amplifying not only the currently known n-damo bacteria but also those that have not been reported, providing new information on the ecological diversity and distribution of this group of microorganisms in the ecosystem. Keywords Anaerobic methane oxidation . pmoA gene . n-damo . PCR primer
Introduction Anaerobic methane oxidizing coupled to denitrification is possible in theory both thermodynamically and biochemically P. Han : J.
