Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR

Table 1 Specific bacterial primers

Genus	Primer(5'→3')
Universal primer	F: CAGGATTAGATACCCTGGTAGT
	R: TTGCGCTCGTTGCGGGACTT
Enterococcus	F: CACCGGAGCTTGCTCCACCG
	R: TGGCTCCAAAAGGTTACTTC
Enterobacterium	F: AGAGCTTGCTCTCGGGTGAC
	R:TAAGCTACCTACTTCTTTTGCAA
Eubacterium	F: GCAACCCTCTCCGGAGGGAAGCG
	R: TTCACCCCCCTCACCCTCCACAC
Bifidobacterium	F: GGCTNGAGCTTGCTCCGGCT
	R: GNCTCACCTTAGACGGCTCC
Lactobacillus acidophilus	F: GAGTTTGATCCTGGCTCAGG
	R: CTGTCCCACCTTAGRCGGCT
Lactobacillus casei	F: GAGTTTGATCCTGGCTCAGG
	R: CTGTCCCACCTTAGRCGGCT

Table 2 Specific probes

Genus	Ligase detection reaction probes	Ligase chain reaction antisense probes	Product length (bp)
Enterococcus	F: (T)20-TTTGACCACTCTAGAGATAG	F: (T)10-TTGCCCCCGAAGGGGAAGCT	80
	R: P-AGCTTCCCCTTCGGGGGCAA(T)20-FAM	R: CTATCTCTAGAGTGGTCAAA-(T)10
Enterobacterium	F: (T)23-TTGGAGGTTGTGCCCTTGAG	F: CTCAAGGGCACAACCTCCAA-(T)10	85
	R: P-GCGTGGCTTCCGGAGCTAAC(T)22-FAM	R: (T)10-GTTAGCTCCGGAAGCCACGC
Eubacterium	F: (T)25-TTGACATATGGGTGAAGCGG	F: CCGCTTCACCCATATGTCAA-(T)10	90
	R: P-GGGAGACCCCGTGGCCGAGA(T)25-FAM	R: (T)10-TCTCGGCCACGGGGTCTCCC
Bifidobacterium	F: (T)28-GGATGTGGGGCCCGTTCCA	F: (T)10-TAGCTCCGACACGGAACCCG	95
	R: P-CGGGTTCCGTGTCGGAGCTAT(T)27-FAM	R: TGGAACGGGCCCCACATCCA-(T)10
Lactobacillus casei	F: (T)30-CAGGTCTTGACATCTTTTGA	F: (T)10-AAACCTGATCTCTCAGGTGA	100
	R: P-TCACCTGAGAGATCAGGTTT(T)30-FAM	R: TCAAAAGATGTCAAGACCTG-(T)10
Lactobacillus acidophilus	F: (T)33-GGTCTTGACATCTAGTGCAA	F: (T)10-GAACTCCGTATCTCTACGGA	105
	R: P-TCCGTAGAGATACGGAGTTC(T)32-FAM	R: TTGCACTAGATGTCAAGACC-(T)10
IC 1#	F: (T)30-CACAGGGCTTTCCACCATCCGTGTC		110
	R: P-GTAGCGGCCAAGCTGCCACGACAGG(T)30-FAM
IC 2#	F: (T)-33-GACATTCGGCAGGCAATCACAGCCT	F: (T)10-GTAAGGTCTTGCAAACGTTCACATC	115
	R: P-GATGTGAACGTTTGCAAGACCTTAC(T)32-FAM	R: AGGCTGTGATTGCCTGCCGAATGTC-(T)10

P: 5’-phosphorylation; FAM : 3’-ends labeled with FAM fluorescence group; IC: Internal control.

Table 3 Design of two sets of probe mixture

Genus	Mixed probes No. 1		Mixed probes No. 2
	LDR (pM)	LCR (pM)	LDR (pM)	LCR (pM)
Enterobacterium	51		5
Eubacterium	5			5
Bifidobacterium	5			5
Enterococcus		5		5
Lactobacillus acidophilus		5		5
Lactobacillus casei		5		5
IC 1#	5		5
IC 2#		5		5

¹The number is the final probe concentration after mixing. LDR: ligase detection reaction; LCR: Ligase chain reaction.

Table 4 Experiment designIand II (detected with probe mixture No. 1 and 2)

Trial	Factor
	Enterobacterium c	Eubacterium	Bifidobacterium	Enterococcus	Lactobacillus casei	Lactobacillus acidophilus	IC 1#	IC 2#
Detected with probe mixture No. 1
1	1	0.5	0.0625	0.01	0.005	0.000625	0.25	0.0025
2	0.5	0.0625	0.125	0.005	0.000625	0.00125	0.0625	0.000625
3	0.25	0.25	0.25	0.0025	0.0025	0.0025	1	0.01
4	0.125	1	0.5	0.00125	0.01	0.005	0.125	0.00125
5	0.0625	0.125	1	0.000625	0.00125	0.1	0.5	0.005
Detected with probe mixture No. 2
1	1	0.005	0.000625	0.01	0.005	0.000625	0.25	0.0025
2	0.5	0.000625	0.00125	0.005	0.000625	0.00125	0.0625	0.000625
3	0.25	0.0025	0.0025	0.0025	0.0025	0.0025	1	0.01
4	0.125	0.01	0.005	0.00125	0.01	0.005	0.125	0.00125
5	0.0625	0.00125	0.01	0.000625	0.00125	0.1	0.5	0.005

“1” is a fixed concentration unit equivalent to 105/μL which was diluted by 104 from standard DNA concentration. IC: internal control.

Table 5 Statistics of sample detection

Bacterium	Enterococcus	Enterobacterium	Eubacterium	Bifidobacterium		Lactobacillus casei	Lactobacillus acidophilus
Probe	LCR	LDR	LDR	LDR	LCR	LCR	LCR
No. of detected sample	29	77	42	46	23	80	77
Detection rate(%)	35.37	93.90	51.22	84.15		97.56	93.90

LDR: Ligase detection reaction; LCR: Ligase chain reaction.

Table 6 Detection limits of mixed samples with ligase detection reaction probes

Eubac	LDR	Dilution times	64	32	16	8	4	2	11	1	1	1	1	1	1
		Detection result	-	+	+	+	+	+	+	+	+	+	+	+	+
IC 1#	LDR	Dilution times	1	1	1	1	1	1	1	2	4	8	16	32	64
		Detection result	+	+	+	+	+	+	+	+	+	+	+	+	+
Bifido	LCR	Dilution times	100	200	400	800	1600	3200	6400	12 800	25 600
		Detection result	+	+	+	+	+	+	+	+	-
IC 1#	LDR	Dilution times	11	1	1	1	1	1	1	1	1
		Detection result	+	+	+	+	+	+	+	+	+
Bifido	LCR	Detecion result	128	64	32	16	8	4	2	12	1	1	1	1	1	1	1
		Dilution times	-	+	+	+	+	+	+	+	+	+	+	+	+	+	+
L. casei	LCR	Detection result	1	1	1	1	1	1	1	1	2	4	8	16	32	64	128
		Dilution times	+	+	+	+	+	+	+	+	+	+	+	+	+	+	-