DNA Terminal-Restriction Fragment Length Polymorphism (T-RFLP)
T-RFLPT-RFLP
In this study, oral microflora was examined in relationship toperiodontitis and malodor, and was further analyzed with febrile days and pneumonia in institutionalized elderly. To characterize the composition of oral microflora, chromosomal DNA was extracted from bacteria in saliva or tongue coat and Terminal-Restriction Fragment Length Polymorphism analysis was carried out using the extracted bacterial DNA as template. The results indicated that T-RFLP pattern of salivary bacteria was closely associated with the prevalence of periodontitis and malodor. In addition, T-RFLP pattern in tongue coat was also associated with febrile days and pneumonia in institutionalized elderly.
T-RFLP
Study of effect of oral microflora on oral and general health
1
九州大学・大学院歯学研究院・教授
1
16S rRNAPCR
Terminal-Restriction Fragment Polymorphism (T-RFLP)
16S rRNA
PCR
T-RFLPT-RFLP
T-RFLP
T-RFLP
T-RFLP
(1)
DNA
6-FMA 8F (5'-AGAGTTTGATYMTGG CTCAG-3')
HEX 806R(5'-GGACTACCR GGGTATCTAA-3')
PCR 16S rRNAHaeIII
T-RFLP T-RFLP
(2)
15 40 200102 98 28.1
T-RFLPI, II, III
(3)
12 74 240102 98 28.1
T-RFLPI, II, III, IV
4
(4)
65
343 85 258 85.8
T-RFLPA, B, C, D
4
37.5
(5)
3400
2612
T-RFLPI, II, III
(1)
III
I II I
FAM19, FAM33, HEX16, HEX22 II
FAM47, HEX32 IIIFAM17. FAM65, HEX12, HEX24
TRFMAW
III
III,
(2)
I
(VSC) VSC
IIIV
Cluster I Cluster II Cluster III (n = 41) (n = 74) (n = 85)
( ) ( SD) 28.9 5.6 28.8 6.6 27.1 6.2 NS b
(%) 14 (34.1) 41 (55.4) 43 (50.5) NS c
(%) 13 (31.7) 16 (21.6) 12 (14.1) NS c
( SD) 0.2 0.6 0.4 1.0 0.2 0.7 NS b
DMFT 9.4 6.4 10.1 7.1 7.8 5.4 NS b
( SD) 27.6 18.1 18.9 19.8 14.6 13.5 I > II, I > III b
( > 4 mm) 10.1 15.4 9.0 15.1 3.7 6.4 I > III, II > III b
10 (24.3) 36 (48.6) 55 (64.7) P < 0.001 c >20% 9 (21.9) 14 (18.9) 2 (2.3) P < 0.001 c
(x 108 deduced CFU per ml) 6.7 7.9 2.6 2.7 1.5 2.0 I > II, I > III ba P values < 0.05 . NS: .
c Fisher's exact test. P values < 0.05 .
a
b Steel-Dwass test. P values < 0.05 .
28.9 5.6
14 (34.1)
13 (31.7)
0.2 0.69.4 6.4
27.6 18.110.1 15.410 (24.3)9 (21.9)6.7 7.9
28.8 6.6
41 (55.4)
16 (21.6)
0.4 1.010.1 7.1
18.9 19.89.0 15.136 (48.6)14 (18.9)2.6 2.7
27.1 6.2
43 (50.5)
12 (14.1)
0.2 0.77.8 5.4
14.6 13.53.7 6.455 (64.7)2 (2.3)
1.5 2.0
T-RFLP
Principle component 1 (40.0%) 0 0.5
Cluster I Cluster II
Cluster III
- 0.5
Cluster I
Cluster II
#17FAM
#24HEX
#65FAM
#32HEX
#33FAM #19FAM
#22HEX
#8HEX
#16HEX
#12HEX #27FAM
#47FAM
Cluster III
VSC (ppm)
(n = 41) (n = 64) (n = 71) (n = 64) (95% CI) (n (%))
T-RFLP Cluster I 21 (51.2) 13 (20.3) 9 (12.7) 0 (0) 1 Cluster II 6 (14.6) 19 (29.7) 33 (46.5) 32 (50.0) 11.1 (5.4-23.2)*** Cluster III 10 (24.4) 22 (34.4) 14 (19.7) 16 (25.0) 5.0 (2.4-10.5)*** Cluster IV 4 (9.8) 10 (15.6) 15 (21.1) 16 (25.0) 10.2 (4.5-23.3)***
*P<0.05, **P<0.01, ***P<0.001.
VSC
II
IV
(3)
H2S CH3SH Cluster I 1 1 Cluster II 10.6 (5.0-22.3)*** 14.6 (6.9-30.8)*** Cluster III 5.9 (2.7-12.7)*** 5.5 (2.5-11.7)*** Cluster IV 16.5 (7.1-38.7)*** 8.4 (3.7-19.0)***
5mm 1 4.9mm 0 0.9mm
N=343
9 = 0; (%) Hazard (%) Odds (95% CI) (95% CI) 1.0 (1.0-1.1) 1.0 (1.0-1.1)
8 (10.7) 1 7 (11.5) 1
27 (10.1) 0.9 (0.4-2.4) 39 (16.4) 0.7 (0.2-2.4)
A 3 (3.0) 1 3 (3.4) 1 B 8 (22.2) 2.7 (0.6-12.6) 15 (11.1) 4.4 (1.1-17.8)* C 13 (8.5) 4.0 (1.1-15.1)* 13 (52.0) 10.5 (2.0-55.5)** D 11 (20.4) 4.9 (1.2-21.1)* 15 (29.4) 11.6 (2.3-57.8)**
or 11 (7.5) 1 17 (12.9) 1 or 24 (12.2) 3.2 (1.4-7.3)** 29 (17.4) 3.7 (1.4-9.6)**
(mm)
1 4.9 14 (10.2) 2.9 (1.1-8.0)* 18 (14.9) 2.7 (1.0-7.3)* 0.9 14 (26.9) 7.9(2.8-22.4)** 16 (39.0) 10.3 (2.9-36.4)**
F29
F68
F53
F32
F56
F90
R29
R20 R13
R30
R39 R10 R43
R12 F36
F42 F57 F80
: Cluster I : Cluster II : Cluster III : Cluster IV
Principal component 1 (45.9%) 0 0.4 -0.4
F29
F56
F90
R30
R12
F42
F32
R20R13
RF80
I II
IV
III Streptococcus Granulicatella Rothia Treponema
Prevotella Veillonella
Fusobacterium Parvimonas Porphyromonas Lautropia Neisseria Haemophilus Aggregatibacter
T-RFLP
Cluster A
Cluster C: Cluster D:
Veillonella
Prevotella Treponema
Streptococcus
Rothia
9 = 0;
(%) Hazard (%) Odds (95% CI) (95% CI)
28 (13.1) 1 37 (20.2) 1
7 (5.4) 1.0 (0.4-2.8) 9 (7.8) 1.5 (0.5-4.0)
3 (3.0) 1 2 (2.3) 1 10 (9.0) 2.6 (0.7-10.1) 9 (8.9) 4.6 (0.8-25.3)
22 (16.8) 1.3 (0.3-6.4) 35 (31.5) 6.7 (1.1-39.4)*
9 (4.8) 1 9 (5.6) 1 9 (8.0) 1.4 (0.5-3.9) 14 (13.5) 1.5 (0.5-4.1) 17 (38.6) 7.0(1.7-28.9)** 23 (67.6) 9.5 (2.1-42.9)**
( ) 0 23 (7.9) 1 33 (12.9) 1 1 - 4 5 (17.2) 2.7 (0.9-8.2) 7 (28.0) 4.0 (1.1-15.1)*
2.8 (1.0-7.5)* 6 (33.3) 3.6 (0.8-15.6)
C, D
Prevotella Treponema Veillonella
Streptococcus Rothia
(4)
I
II III
Neisseria HaemophilusI
II,IIIPrevotella
Veillonella StreptococusRothia
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