Table 2.

PCR primers and probes used for the quantification of ruminal protozoal and methanogenic microbial populations by qPCR analysis.

Target	Primer/Probe Name and Sequence	Assay	Efficiency (%)	Reference
Total bacteria (rrs)	V3-16S-F, 5′-CCTACGGGAGGCAGCAG-3′ V3-16S-R, 5′-ATTACCGCGGCTGCTGG-3′	SYBR	96	[56]
Total methanogens (rrs)	Met630F, 5′-GGATTAGATACCCSGGTAGT-3′	SYBR	92	[57]
	Met803R, 5′-GTTGARTCCAATTAAACCGCA-3′
Total prokaryotes (rrs; reference gene)	V3-F, 5′-CCTACGGGAGGCAGCAG-3′	SYBR	91	[56]
	V3-R, 5′-ATTACCGCGGCTGCTGG-3′
M. stadtmanae (rrs)	Stad-F, 5′-CTTAACTATAAGAATTGCTGG-3′	SYBR	98	[58]
	Stad-R, 5′-TTCGTTACTCACCGTCAAGAT-3′
M. smithii (rrs)	Smit.16S-740F, 5′-CCGGGTATCTAATCCGGTTC-3′	FAM 1	91	[59]
	Smit.16S-862R, 5′-TCCCAGGGTAGAGGTGAAA-3′
	Smit.16S FAM, 5′CGTCAGAATCGTTCCAGTCA-3′
M. ruminantium (rrs)	Rum16S 740F, 5′-TCCCAGGGTAGAGGTGAAA-3′	FAM	92	[28]
	Rum16S 862R, 5′CGTCAGAATCGTTCCAGTCA-3′
	Rum16S FAM, 5′-CCGTCAGGTTCGTTCCAGTTAG-3′
Protozoa	Prot 18S F 5′-GCTTTCGWTGGTAGTGTATT-3′	SYBR	95	[54]
	Prot 18S R 5′-CTTGCCCTCYAATCGTWCT-3′

¹ SYBR = Fast SYBR Green I Dye assay; FAM = TaqMan probe-based assay.