Discovery of a novel rumen methanogen in the anaerobic fungal culture and its distribution in the rumen as revealed by real-time PCR

Table 3 Primers used in this study to target 16S r RNA genes of total archaea and the novel RCC species, mcrA genes of methanogens

Target	Primers	Sequence (5’-3’)	Annealing temp (°C)	References
Archaea*	915f 1386r	GTGCTCCCCCGCCAATTCCT GCGGTGTGTGCAAGGAGC	59	[11]
Methanogen	86f	GCTCAGTAACACGTGG	56	[2]
Methanogen	1340r	CGGTGTGTGCAAGGAG	56	[2]
The novel RCC species*	178f	TGGGATCTGGAATGACCCATGG	56	This study
The novel RCC species*	434r	TGAGAAAAGCTAGAACAAATGTCCT	56	This study
Methanogen^※	519f	CAGCCGCCGCGGTAA	57	[39]
Methanogen^※	915r^#	CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCG	57	[39]
Methanogen (mcr A gene)	MLf	GGTGGTGTMGGATTCACACARTAYGCWACAGC	55	[40]
	MLr	TTCATTGCRTAGTTWGGRTAGTT

*, For real-time PCR; ^※, For DGGE; ^#, Where the primer name is prefaced by ‘GC-’ in the text, a GC-clamp was added to the 5’.
-terminus of the primer. The GC-clamp sequence was CCCCGTGCTCCCCCGCCAATTCCT;.

ISSN: 1471-2180