Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification

Table 2 Phylum level classification, primers and assay conditions of qPCR assays

qPCR assay (Phylum)	Primers (5’→3’)	Standard	Target size (bp)	MgCl2 (mmol/L)	Annealing T (°C)	Detection T (°C)	Average PCR efficiency ± SD
Bacteroides intestinalis-like (Bacteroidetes)	F: AGCATGACCTAGCAATAGGTT	AM277809	124	3	63	83	87 ± 6
	R: CCTTCTCGTTATACTATCCGGTAT
Bifidobacterium catenulatum/Bifidobacterium pseudocatenulatum-like[11] (Actinobacteria)	F: ACTCCTCGCATGGGGTGTC	AM277149	275	3	68	87	90 ± 8
	R: CCGAAGGCTTGCTCCCGAT
Butyrivibrio crossotus-like (Firmicutes)	F: TGCTAATACCGCATAAAACAGCAGA	AM275497	232	4	63	85	82 ± 5
	R: CGCTGGATCAGGCTTTCG
Clostridium cocleatum 88%[11] (Firmicutes)	F: AATACATAAGTAACCTGGCRTC	AM276544	104	4	60	80	88 ± 10
	R: CGTAGCACTTTTCATATAGAGTT
Clostridium thermosuccinogenes 85% (Firmicutes)	F: ACATGCAAGTCGAACGGAAGTC	AM275406	373	2	62	81	88 ± 8
	R: TGCGTCAGAGTTTCCTCCATTG
Collinsella aerofacien-like[11] (Actinobacteria)	F: CCCGACGGGAGGGGAT	AM276090	260	4	67	89	75 ± 3
	R: CTTCTGCAGGTACAGTCTTGAC
Coprobacillus catenaformis 91% (Firmicutes)	F: CGGACGCGATGCTTCT(A/G)GC	AM275478	133	4	62	82	93 ± 8
	R: AACATATCTCCCATGCGGTTG
Coprococcus eutactus 97%[11] (Firmicutes)	F: AGCTTGCTCCGGCYGATTTA	AM275825	97	2	63	83	73 ± 5
	R: CGGTTTTACCAGTCGTTTCCAA
Ruminococcus bromii-like (Firmicutes)	F: CGAACGGAACTGTTTTGAAAGA	AM275413	156	4	62	81	97 ± 6
	R: CAAAACCATGTGGTTCCGATAT
Ruminococcus torques 91%[11] (Firmicutes)	F: TGCTTAACTGATCTTCTTCGGA	AM276624	119	5	62	82	88 ± 5
	R: CGGTATTAGCAGTCATTTCTG
R. torques 93%1(Firmicutes)	F: GACTGCTTTTGAAACTGTCA	AM275798	396	4	61	83	85 ± 4
	R: AGGTCCGGTTAAGGA
R. torques 94%[11] (Firmicutes)	F: AATCTTCGGAGGAAGAGGACA	AM275522	137	2	65	85	81 ± 6
	R: ACACTACACCATGCGGTCCT
Slackia faecicanis 91% (Actinobacteria)	F: GAGTAACGCGTGACCGACCTT	AM276086	75	4	64	86	90 ± 5
	R: CCCGGAGTACCCGGTATCA
Spiroplasma chinense 84% (Firmicutes)	F: ATGGCCCAGTGAAGGTTG	AM275518	101	4	66	83	79 ± 4
	R: CCCAACGAAAAGGTAGGTCA
Universal[61]	F: TCCTACGGGAGGCAGCAGT	B. longum2	466	3	50	80	92 ± 6
	R: GGACTACCAGGGTATCTAATCCTGTT

¹For the R. torques 93% assay the sequence AY305319[62] was used for primer design;

²The Bifidobacterium longum DSM 20219T 16S rRNA gene was used as standard in the universal qPCR assay. qPCR: Quantitative real-time polymerase chain reaction.