Table 1.

Target-specific primer sequences constituting the Gut Microbiotassay in order to target main bacterial phyla and bacterial groups and species of high interest in the mammalian intestine

Main target		rRNA gene	Sequence (5′ → 3′)	E. coli position	Size, bp	Reference	Reference bacterium
Domain Bacteria A V2-V3	Forward	16S	AGAGTTTGATCCTGGCTCAG	7	336	Liu et al.[30]	1,2,3,4,5,6,7,8,9,10,11,13,14,15
	Reverse	16S	CTGCTGCCTYCCGTA	342		Liu et al.[30]
Domain Bacteria B V4-V5	Forward	16S	CAGCAGCCGCGGTAATAC	518	389	Schwieger & Tebbe [31]	1,2,3,4,5,6,7,8,9,10,11,13,14,15
	Reverse	16S	CCGTCAATTCCTTTGAGTTT	906		Schwieger & Tebbe [31]
Phylum Firmicutes	Forward	16S	CTGATGGAGCAACGCCGCGT	385	429	Haakensen et al.[32]	6,7,9,11,13,14
	Reverse	16S	ACACYTAGYACTCATCGTTT	813		Mühling et al.[33]
Class Bacilli	Forward	16S	GCAGTAGGGAATCTTCCGC	353	461	Felske et al.[34]	7,11,14
	Reverse	16S	ACACTTAGCACTCATCGTTT	813		Modified from Mühling et al.[33]
Genus Enterococcus	Forward	16S	GGGTAACCTRCCCATCAGA	125	325	Modified from Behr et al.[35]	7
	Reverse*	16S 16S	GTTACTCTCATCCTTGTTC ACCGTCAGGGGACGTTCAG	449 466	342	Modified from Behr et al.[35] Modified from Behr et al.[35]
Genus Lactobacillus	Forward	23S	GCGGTGAAATTCCAAACG	774	216	This study, Linux ARB [13]	11
	Reverse	23S	GGGACCTTAACTGGTGAT	989		This study, Linux ARB [13]
Genus Streptococcus	Forward	16S	CTWACCAGAAAGGGACGGCT	488	337	This study, ClustalW2 [36]	14
	Reverse	16S	AAGGRYCYAACACCTAGC	824		This study, ClustalW2 [36]
Family Clostridium cluster I	Forward	16S	AAAGGAAGATTAATACCGCATA	159	538	Modified from Rinttila et al.[37]	6
	Reverse	16S	TTCTTCCTAATCTCTACGCA	696		Hung et al.[38]
Species Clostridium perfringens	Forward	16S	TGAAAGATGGCATCATCATTCAAC	183	258	Skånseng et al.[39]	6
	Reverse	16S	GGTACCGTCATTATCTTCCCCAAA	440		Skånseng et al.[39]
Family Clostridium cluster IV	Forward	16S	ACAATAAGTAATCCACCTGG	866	298	Modified from Ramirez-Farias et al.[40]	9
	Reverse	16S	CTTCCTCCGTTTTGTCAA	1163		Matsuki et al.[41]
Family Clostridium cluster XIV	Forward	16S	CGGTACCTGACTAAGAAGC	482	413	Rinttila et al.[37]	13
	Reverse	16S	CTTTGAGTTTCATTCTTGCGAA	894		Matsuki et al.[42]
Phylum Bacteroidetes	Forward	16S	CCGGAWTYATTGGGTTTAAAGGG	554	414	Mühling et al.[33]	1
	Reverse	16S	GGTAAGGTTCCTCGCGTA	967		Mühling et al.[33]
Genus Bacteroides	Forward	16S	AAGGTCCCCCACATTGG	302	300	Manz et al.[43]	1
	Reverse	16S	GAGCCGCAAACTTTCACAA	601		Franks et al.[44]
Phylum Actinobacteria	Forward	16S	GCGKCCTATCAGCTTGTT	235	333	Modified from Glockner et al.[45] first published. They refer to Erhart [46]	2
	Reverse	16S	CCGCCTACGAGCYCTTTACGC	567		This study, ClustalW2 [36]
Family Bifidobacteriaceae	Forward	16S	CTCCTGGAAACGGGTGG	152	442	Matsuki et al.[42]	2
	Reverse	16S	CTTTCACACCRGACGCG	593		Delroisse et al.[47]
Class β- and γ-proteobacteria	Forward	23S	GTATAATGGGTCAGCGAC	569	673	This study, Linux ARB [13]	8
	Reverse	23S	CAGCATTCGCACTTCTGA	1241		This study, Linux ARB [13]
Family Enterobacteriacea	Forward	16S	CGTCGCAAGMMCAAAGAG	182	333	Modified from Friedrich et al.[48]	8
	Reverse	16S	TTACCGCGGCTGCTGGCAC	514		Modified from Palmer et al.[49]
Species Escherichia coli	Forward	16S	GTTAATACCTTTGCTCATTGA	461	320	Malinen et al.[50]	8
	Reverse	16S	ACCAGGGTATCTAATCCTGTT	780		Malinen et al.[50]
Class ϵ-proteobacteria	Forward	16S	TGGTGTAGGGGTAAAATCCG	680	286	Bui et al.[51]	5
	Reverse	16S	AGGTAAGGTTCTTCGYGTATC	965		This study, Primrose [14]
Class δ-proteobacteria	Forward	16S	GGTGTAGGAGTGAARTCCGT	681	534	This study, Primrose [14]	3
	Reverse	16S	TACGTGTGTAGCCCTRGRC	1214		This study, Primrose [14]
Phylum Fusobacteria	Forward	16S	GATCCAGCAATTCTGTGTGC	387	292	Sanguin et al.[52]	10
	Reverse	16S	CGAATTTCACCTCTACACTTGT	678		Walter et al.[53]
Phylum Verrucomicrobia	Forward	16S	GAATTCTCGGTGTAGCA	673	551	Modified from Ranjan [54]	15
	Reverse	16S	GGCATTGTAGTACGTGTGCA	1223		This study, Primrose [14]
Phylum Spirochaetes	Forward	16S	GTYTTAAGCATGCAAGTC	45	294	Choi et al.[55]	4
	Reverse	16S	TGCTGCCTCCCGTAGGAG	338		This study, ClustalW2 [36]
Domain Archaea	Forward	16S	CAGCMGCCGCGGTAATWC	518	440	Giovannoni et al.[56]	12
	Reverse	16S	YCCGGCGTTGAMTCCAATT	957		Delong [57]

Reference bacteria listed are target for the respective primer sets. Numbers represent: 1: Bacteroides fragilis (DJF_B083(EU728706)), 2: Bifidobacterium pseudolongum globosum (DMS 20092), 3: Bilophila wadsworthia (ATCC 49260), 4: Brachyspira pilosicoli (isolated from the intestine of slaughter pig at DTU-VET, 28-02-2000), 5: Campylobacter fetus (ATTC 10852), 6: Clostridium perfringens (NCTC 10240), 7: Enterococcus faecalis (ATCC 29212), 8: Escherichia coli (9711108–2), 9: Faecalibacterium prausnitzii (DSM 17677), 10: Fusobacterium Necrophorum (ATCC 25286), 11: Lactobacillus sakei (DSM 20017), 12: Methanocorpusculum labreanum (DSM 4855), 13: Roseburia sp. (DJF_VR77(EU728794)), 14: Streptococcus suis (NCTC 10446), and 15: Verrucomicrobium spinosum (DSM 4136). Nucleotide explanation: Y, C/T ; R, A/G ; W, A/T ; K, G/T ; M, A/C.

*To improve coverage of the Genus Enterococcus, two reverse primers were mixed in equal concentrations.