Professional Documents
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B Y T
TRNG I HC DC H NI
LUN VN THC S DC HC
H NI 2014
B GIO DC V O TO
B Y T
TRNG I HC DC H NI
H NI 2014
LI CM N
Lun vn ny c hon thnh ti khoa Vi Sinh Vin Kim nghim Thuc
Trung ng B Y t. c c bn lun vn tt nghip ny ti xin by t lng
bit n chn thnh v su sc ti TS. Trn Vit Hng, ngi thy trc tip hng
dn, du dt ti trong sut thi gian thc hin nghin cu ny.
Xin chn thnh cm n cc thy c trong Ban Gim hiu, Phng o to sau
i hc, B mn Kim Nghim Thuc v c cht v cc B mn khc ca trng
i Hc Dc H Ni gip ti trang b tri thc, to mi trng, iu kin thun
li cho ti trong sut qu trnh hc tp ti trng cng nh trong qu trnh hon
thnh lun vn.
Ti xin chn thnh cm n TS. on Cao Sn Vin trng Vin kim
Nghim Thuc Trung ng, Ban Gim c Vin Kim Nghim Thuc Trung
ng, cc ng nghip trong khoa v Th s. Khng Th Minh Hu h tr, gip
ti hon thnh tt lun vn thc s v hon thnh kha hc theo ng thi hn.
Cui cng, ti xin gi li tri n su sc n gia nh v bn b lun bn
ti ng vin, chia s, gip ti trong thi gian va qua
H Ni 27 thng 8 nm 2014
Dc s Trnh Th Phng Dung
MC LC
CC CH VIT TT
DANH MC BNG
DANH MC HNH
T VN ....................................................................................................... 1
CHNG 1. TNG QUAN ................................................................................ 3
1.1. TNG QUAN V PROBIOTICS ............................................................... 3
1.1.1. nh ngha ................................................................................................ 3
1.1.2. ng dng ca probiotics trong ngnh Dc ............................................ 3
1.1.3. Cc vi sinh vt thng c s dng trong ch phm probiotics............ 4
1.1.4. Tng quan v tiu chun cht lng probiotics ....................................... 4
1.2. TNG QUAN V CHI BIFIDOBACTERIUM .......................................... 6
1.2.1. c im chung ca chi Bifidobacterium [30], [33],............................... 6
1.2.2. Bifidobacteria vi vai tr l probiotics. ................................................... 6
1.3. TNG QUAN V B. LONGUM .................................................................. 7
1.3.1. c im hnh thi ................................................................................... 7
1.3.2. Kh nng chuyn ha carbohydrat [31]. .................................................. 8
1.3.3. Cu trc gen cu B. longum [24].............................................................. 8
1.3.4. Vai tr ca B. longum [9], [11], [24], [26] , [15], [33], [35]. ................... 9
1.3.5. Phng php kim nghim B. longum trong ch phm probiotics ........10
1.3.6. Tnh hnh nghin cu nh tnh v nh lng B. longum trong cc ch
phm probiotics trong nhng nm gn y ...........................................17
CHNG 2. I TNG, NGUYN VT LIU, TRANG THIT B
PHNG PHP NGHIN CU .............................................................20
2.1. PHNG TIN DNG TRONG NGHIN CU ..................................20
2.1.1. Mi trng, ho cht, dung mi, thit b ...............................................20
2.1.1.1. Mi trng ......................................................................................20
2.1.1.2. Ho cht, dung mi .........................................................................22
2.1.1.3. Thit b, dng c .............................................................................23
2.1.2. Phn mm x l kt qu: Apiweb ca hng BioMrieux. ......................24
2.1.3. Chng vi sinh vt chun .........................................................................24
2.2. I TNG NGHIN CU ....................................................................25
CC CH VIT TT
AA:
Acid acetic
AL:
Acid lactic
ATCC:
B. longum:
Bifidobacterium longum
dATP:
dCTP:
Deoxy cytosineTriphosphat
dGTP:
DNA:
dNTP:
dTTP:
E. coli :
Escherichia coli
EDTA
Ethylenediaminetetraacetic acid
FAO
FISH:
JCM:
LAB:
LB
Luria Bertani
PCR:
rRNA:
VSV:
Vi sinh vt
DANH MC BNG
Trang
Bng 2.1
Mi c hiu
22
Bng 2.2
Ha cht, dung mi
22
Bng 2.3
24
Bng 2.4
30
Bng 2.5
34
Bng 2.6
35
Bng 2.7
36
Bng 2.8
38
Bng 2.9
40
agar
Bng 2.10
42
Bng 2.11
43
Bng 3.1
45
Bng 3.2
48
Bng 3.3
50
Bng 3.4
51
Bng 3.5
53
Bng 3.6
62
Bng 3.7
64
Bng 3.8
66
20 chng
Bng 3.9
68
Bng 3.10
69
DANH MC HNH
Trang
Hnh 1.1
Hnh 1.2
Hnh 1.3
14
Hnh 1.4
14
Hnh 2.1
31
Hnh 3.1
46
Hnh 3.2
46
Hnh 3.3
47
Hnh 3.4
47
Hnh 3.5
47
Hnh 3.6
48
Hnh 3.7
52
54
55
56
Hnh 3.11
57
58
59
Hnh 3.14
63
Hnh 3.15
65
Hnh 3.16
67
70
T VN
Vi khun lactic (LAB) c vai tr rt quan trng trong cuc sng ca chng ta.
Chng to ra cc thc phm ln men v bo qun thc phm khi b h hng. T
u th k 20, Elie Metchnikoff (1845-1916) xut s dng cc LAB cho mc
ch cha bnh. T , lnh vc nghin cu probiotics ngy cng c quan tm v
pht trin. n nay, nhng nghin cu v probiotics khng ngng cung cp
nhng bng chng c tnh khoa hc v hiu qu thc s ca probiotics i vi sc
khe con ngi. Bn cnh , cc sn phm chc nng s dng cc vi khun
probiotics xut hin ngy cng nhiu Chu u, Nht, M Nm 2008, doanh thu
t cc ch phm ny l 15,9 t USD trn ton cu [10].
Probiotics bt ngun t ngn ng Hy Lp c ngha l v s sng (for life).
Nm 2002 t chc Nng Lng Lin hip quc v t chc Y t th gii nu r:
probiotics l nhng vi sinh vt sng m khi s dng vi mt lng thch hp, s
to nn nhng hiu qu tt i vi sc khe ca c th ch [32]. Cc probiotics
thng c trong cc ch phm l vi khun sinh acid lactic (LAB) trong bao gm
chi Lactobacillus v chi Bifidobacterium, vi khun sinh bo t Bacillus, nm
Saccharomyces cerevisiae
Theo Robin Temmerman (2001) trong s 55 mu kho st ch c khong 20%
ch phm c cha vi khun ng nh trn nhn, 16% ch phm khng cha mt
trong cc chng probiotics c lit k trn nhn [28]. Mt khc s lng vi sinh
vt trong cc ch phm probiotics gim nhiu trong thi gian bo qun. Nh vy,
vic nh tnh, nh lng cc loi vi khun probiotics trong cc ch phm l rt
quan trng, m bo cht lng ca ch phm, mang li hiu qu cho ngi dng.
Th trng thuc Vit Nam hin c hng trm ch phm probiotics di dng
thuc v thc phm chc nng, trong c rt nhiu ch phm c cha
Bifidobacterium longum (B. longum l mt vi khun thuc nhm LAB) di dng
n hoc a thnh phn. Mc d trn th gii c rt nhiu cng trnh nghin cu
nh tnh v nh lng B. longum trong cc ch phm a thnh phn, tuy nhin
Vit Nam vic kim sot cht lng ca cc ch phm probiotics cn ang b
ng. C th l Vit Nam cha c mt ti liu chnh thc no hng dn kim sot
cht lng cc ch phm probiotics bao gm c cc ch phm cha B. longum. a
s cc tiu chun c s ca cc thuc v thc phm chc nng trong nc cng nh
nhp khu cha nh lng ring cng nh cha nh tnh c B. longum trong
cc ch phm c cha hn hp nhiu vi sinh vt.
Xut pht t nhu cu thc t v tnh cp thit v yu cu kim tra cht lng
ca cc ch phm probiotics, chng ti tin hnh ti: Xy dng quy trnh xc
nh (nh tnh, nh lng) Bifidobacterium longum trong cc ch phm
probiotics vi cc mc tiu:
- Xy dng quy trnh chun nh tnh, nh lng B. longum trong cc ch
phm a thnh phn.
- ng dng quy trnh thit lp kho st cht lng ca mt s ch
phm probiotics c cha B. longum ang lu hnh trn th trng.
- Cc yu cu v nhn sn phm
Nm 2009, B y t Canada cng ban hnh chuyn mc v probiotics, trong
cng yu cu nh danh vi sinh vt n loi bao gm c kiu hnh v kiu gen [10].
n nm 2011, B sc khe v gia nh cng vi B Cng ngh sinh hc ca
n ban hnh hng dn nh gi cht lng probiotics kh y bao gm
9 ch tiu [16]:
- nh danh vi sinh vt: yu cu phi nh danh n loi bao gm c kiu
hnh v kiu gen. Trong ch ra k thut PCR l k thut sinh hc phn t
nh danh vi sinh vt.
- Cc th nghim in vitro v hot tnh ca probiotics: khng khng sinh,
khng dch v, khng mui mt
- Cc bng chng v s an ton trn ng vt
- Cc th nghim v tc dng ca probiotics trn ng vt
- Cc bng chng v s an ton trn ngi
- Th tc dng trn ngi
- Cc yu cu v nhn sn phm
- Cc th nghim xc nh liu
- Yu cu ghi nhn
- Yu cu trong sn xut v th tc ng k
Hin nay Vit Nam cha c vn bn chnh thc no v vic nh gi cht
lng ca cc ch phm probiotics. Ch c duy nht TCVN 7849:2008 a ra
phng php m L. acidophilus trong sa v cc ch phm sa trn mi trng
c hiu [5]. Vic kim sot cht lng mi ch dng li xc nh s lng vi sinh
vt, nh tnh da trn kiu hnh, nhim khun v cc ch tiu v dng bo ch
theo tiu chun nh sn xut. Trong rt nhiu trng hp, tiu chun cht lng cn
rt s si, c bit cha nh danh c tt c cc vi sinh vt trong ch phm.
Trong :
A: Kch thc gen biu din trn thang Mb
B: Chui m ha: gm 2 si b sung, cc mi tn ch phn chuyn tip GC
C: Thang G-C
D: Cu to nguyn vn genom ca B. longum.Vng hnh trn biu din cc
rARN operon, hnh vung l vng gen ca prophage, tam gic biu din v
tr ca 3 vng gen ging nhau ca B. longum, hnh ch nht l vng gen
plasmid tch hp.
1.3.4. Vai tr ca B. longum [9], [11], [24], [26] , [15], [33], [35].
B. longum l mt loi vi khun rt cn thit vi c th con ngi, thng gp
rut v m o do n c vai tr quan trng trong vic gi cho h tiu ha v h
min dch con ngi khe mnh. B. longum c bit quan trng i vi tr s sinh
do n gip tng cng h min dch ca tr s sinh. B. longum c nhiu trong h
tiu ha ca tr s sinh v chim u th hn tr b m so vi tr b bnh. Mt
iu d thy l tr b m t b tiu chy v d ng hn so vi tr b bnh. ngi
ln, B. longum gi cho h thng min dch khe mnh, h tr cn bng vi khun
ng rut v c th ngn nga ung th nh ung th rut kt. B. longum sn sinh
acid lactic, do vy lm gim nguy c vim m o.
Mt nghin cu lin quan n gim cn, bo ph v to bn ch ra rng nhng
ngi s dng B. longum t c kt qu gim cn v ci thin to bn tt hn so
vi nhng ngi dng thuc nhun trng [11].
Mt nghin cu khc hin c thc hin ti Nht Bn nhm xc nh liu B.
longum c th c s dng nh l vector chuyn gen iu tr ung th hay
khng. Tin hnh thc nghim trn chut cho thy B. longum c th xm nhp vo
khi u v tch ly trong khi u. y l mt nghin cu rt tch cc v hy vng n s
gip cho cc nh nghin cu tm ra cch mi a thuc trc tip vo khi u [15].
Do c vai tr rt quan trng i vi sc khe con ngi nn B. longum c
s dng rng ri trong cc ch phm thuc, thc phm chc nng, sa...
10
v pht tn hiu hunh quang mnh m hn (so vi trng thi t do trong dung
dch). Loi tc nhn hunh quang ny c th s dng c cho mi trnh t ch
nn chi ph s thp; nhng c hiu ca sn phm phi c kim tra cht ch
thng qua mt bc phn tch b sung sau khi phn ng nhn bn kt thc phn
tch ng cong nng chy (Melting curve analysis).
Tc nhn dng nh du mu d c hiu rt a dng nh FAM, TAMRA,
Texas Red, Cy3, Cy5 c s dng vi nhiu loi mu d khc nhau nh mu d
lai, mu d thy gii, mu d dng kp tc.
Khi s dng vi cc mu d khc nhau, cng tn hiu hunh quang u t
l thun vi hm lng sn phm ch ang c nhn bn trong phn ng. Cc tn
hiu hunh quang pht ra s c cc u d ca my lun nhit real-time PCR thu
nhn v x l. Khi cng tn hiu hunh quang ca mu vt qua ng tn hiu
hunh quang nn (base line) ca phn ng th mu c xem l dng tnh v
ngi ta ly thi im vt qua (biu hin qua gi tr chu k ngng Ct, Cycle
Threshhold) so snh vi mt ng cong chun (standard curve) bit suy
ra nng trnh t DNA ch trong mu th nghim ban u. ng cong chun
c xy dng t chu k ngng ca cc mu chun c cha sn phm ch bit
trc nng .
c. Phng php lai hunh quang ti ch (FISH-fluorescent insitu
hybridization).
FISH d tm v pht hin ra cc trnh t acid nucleic ca vi sinh vt cn nh
lng bng cch dng u d c nh du hunh quang lai c hiu vi cc trnh
t c hiu b sung vi n bn trong t bo nguyn vn (khng cn ph v t bo)
Quy trnh thc hin bao gm cc bc:
- Chun b mu v u
- C nh mu
- Lai gia u d v trnh t ch c hiu nm trong t bo
- Ra mu loi b cc u d khng c lai
- D tm mu (bc ny c th b qua nu s dng u d pht hunh quang
trc tip)
11
12
13
Hnh 1.4.
14
15
- Nhn dng
Nhn dng l s tch lp v thu nhn nhiu bn sao ng nht ca mt gen
hay ca mt mnh on DNA.
Nguyn tc:
K thut nhn dng bao gm vic phi ci mt mnh (chui) DNA l vo mt
vector (plasmide hoc phage ) bng phng php ha sinh. Sau , a phn t
lai nay vo t bo ch chn la bng phng php bin np hoc ti np. Trng
hp mun to dng tng hp enzym th mnh DNA nh ci phi m ha cho gen
cu trc ca mt enzyme no .
+ Cc bc c bn ca phng php nhn dng:
Tch lp cc DNA l cn nhn dng:
Chn v x l vector nhn dng
To DNA ti t hp (vector ti t hp).
Chuyn DNA ti t hp vo t bo ch bng phng php bin np hoc ti
np.
Pht hin dng cn tm
Kim tra thu nhn sn phm ca gen ti t hp.
- Gii trnh t
Phng php chnh xc v a li nhiu thng tin nht cho nh danh v nh
typ vi sinh vt l xc nh chnh xc trnh t chui DNA ca mt vng quy nh
trn nhim sc th. Phng php gii trnh t DNA pht trin nhanh chng, kt qu
so snh s tng ng ca trnh t gen nghin cu tr thnh phng php phn loi
chun theo sinh hc phn t trong nghin cu h thng hc v cy ph h di truyn
gia cc i tng nghin cu. Cc gen bo th c gii trnh t lm c s xc
nh v tr ca sinh vt nghin cu, trong khi cc trnh t khng tng ng
(khc bit) li lm c s cho s bit ha cng nh xc nh mi quan h gia cc c
th gn vi nhau.
T nhng nm 1977 mt s phng php gii trnh t gen c pht minh
nh phng php gii trnh t gen theo phng php ha hc (Alan
16
Maxam v Walter Gilbert), phng php gii trnh t gen bng enzyme (do
Frederick Sanger v cng s )
C hai phng php ny nh du bc ngot ln trong lch s pht trin
ca b mn sinh hc hin i. Ngy nay, rt nhiu my gii trnh t gen t ng u
da trn nguyn tc chnh l phng php gii trnh t gen ca Sanger.
Phng php ny da vo s tng hp mch b sung cho trnh t cn xc nh
nh DNA polymerase. Vi vic s dng thm cc dideoxynucleotide cng vi cc
deoxynucleotid thng thng, kt qu tng hp cng l s hnh thnh mt tp hp
nhiu on DNA c kch thc khc nhau.
Cc phn on DNA s c phn tch bng in di trn gel polyacrylamid c
kh nng phn tch hai trnh t DNA ch chnh nhau mt nucleotid. Vi vic s
dng mt loi nucleotide c nh du ng v phng x, kt qu trnh t DNA cn
xc nh c c trn bn phng x t ghi t bn in di.
thc hin c gii trnh t bng my t ng th cc mch DNA n sn
sinh trong ng phn ng gii trnh t phi c nh du hunh quang cc vch
in di ca cc mch n ny pht sng khi i qua mt chm tia sng laser. Nguyn
tc hot ng ca my l trong sut qu trnh in di, mi khi c mt vch in di
i qua chm tia laser th vch in di s pht sng ln v s pht sng ny s c
con mt cn quang ghi nhn v lu li thnh mt nh cng sng trong biu .
T biu ca cc nh cng sng ny, my s so dng ca cc nh tng ng
vi cc mu cui cng phn tch thnh trnh t ca on DNA.
17
18
19
10,0g
Cao tht
8,0g
4,0g
Glucose
20,0g
Dikali hydrophosphat
2,0g
Tween 80
1,0g
Amoni citrat
2,0g
Natri acetat
5,0g
Mangan sulfat
0,04g
Magnesi sulfat
0,2g
Thch
14,0g
Nc ct va
1000ml
20
5,0g
5,0g
Natri clorid
2,5g
L-tryptophan
0,2g
L-cystine
0,4g
Hemin
0,005g
Vitamin K1
0,01g
Natri sulfit
0,1g
Nc ct va
1000ml
10,0g
Natri clorid
5,0g
Cao tht
2,5g
Thch
20,0g
Nc ct va
1000ml
7,4 7,6
50,0ml ( mu 5%)
21
B. longum
Tn mi
S l
Hng sn xut
ID16R08F
208918A07
IDL16R09R
208918A08
BlonF
208918B05
BlonR
208918B05
Invitrogen
Invitrogen
Promega
S l
Hng sn xut
Lysozym
061M13281V
Sigma-Aldorich
297885
Promega
1130853
Life technologies
dNTPs (100mM)
1139131
Life technologies
668361
Invitrogen
00056239
Fermentas
1113366
Invitrogen
764952
Invitrogen
0000144547
Invitrogen
90920S196
Biobasic Inc
1004SHLS0125B0210J
Biobasic Inc
1002998430
BioMrieux
Thuc th BCP
1003101450
BioMrieux
Purification Kit
Thuc th XYL
Thuc th HER
Merck
1003059340
22
BioMrieux
23
Tn chng
K hiu
TT
Tn chng
K hiu
Bacillus
ATCC 11778
11
Lactobacillus
ATCC
rhamnosus
9595
Bifidobacterium
ATCC
breve
15700
Bifidobacterium
ATCC
bifidum
11863
Streptococcus
ATCC
faecalis
33186
Streptococcus
ATCC
pyogenes
19615
Pseudomonas
ATCC
aeruginosa
9027
Escherichia coli
ATCC
cereus
2
Bacillus
ATCC 14884
12
pumilus
3
Bacillus
ATCC 795
13
stearothermo
philis
4
Bacillus
ATCC 6333
14
subtilis
Lactobacillus
ATCC
acidophilus
4356
Lactobacillus
ATCC 7830
15
16
leichmannii
7
Bacilus
ATCC 7050
17
coagulans
8
Lactobacillus
8739
ATCC 334
18
casei
9
Lactobacillus
ATCC 9338
19
fermentum
10
Lactobacillus
ATCC 8014
20
plantarum
Staphylococcus
ATCC653
aureus
Streptococcus
ATCC
thermophilus
19258
Bifidobacterium
JCM 1217
longum
24
108CFU
+ Bifidobacterium longum
108CFU
+ Streptococcus faecalis
108CFU
108CFU
+ Bifidobacterium longum
108CFU
+ Streptococcus thermophilus
108CFU
- Gi bt L-Bio-3D (k hiu mu C)
Cng ty sn xut: Cng ty lin doanh dc phm MEBIPHARAUSTRAPHARM
S ng k: QLSP -0746-13
S l: 061213. Hn dng: 25/12/2015
Cng thc: Mi gi 1g bt cha hn hp cc vi khun sinh acid lactic
108CFU
+ Lactobacillus acidophilus
+ Bifidobacterium longum
+ Lactobacillus rhamnosus
- Gi bt Lackid (K hiu mu D)
25
26
37
MTB
Trong :
A
nTB
MTB
27
28
29
Hm
Ging
Cht nn
lng(mg/gi
Kt qu
Phn ng
Dng tnh
m tnh
ng)
L-tryptophan
0,98
Vng
URE
Urea
0,648
Urease
Vng-cam
BCP
GLU
D-glucose
1,96
Acid ha
MAN
D-manitol
1,96
Acid ha
LAC
D-lactose
1,96
Acid ha
SAC
D-saccharose
1,86
Acid ha
MAL
D-maltose
1,96
Acid ha
SAL
Salicin
1,64
Acid ha
XYL
D-xylose
1,64
Acid ha
ARA
L-arabinose
1,64
Acid ha
GEL
gelatin
0,6
ESC
Esculin
0,36
St (III) citrat
0,11
Tm
Vng/ vng-xanh
C s khuch tn
mu
cht mu en
Vng
Nu-en
Soi di n UV 365nm
S thy phn Esculin
Pht quang
GLY
Glycerol
1,82
Acid ha
CEL
D-cellobiose
1,86
Acid ha
MNE
D-mannose
1,96
Acid ha
MLZ
D-melezitose
1,96
Acid ha
RAF
D-raffinose
2,18
Acid ha
SOR
D-sorbitol
2,18
Acid ha
RHA
L-rhamnose
1,96
Acid ha
TRE
D-trehalose
1,96
Acid ha
Tm
Vng/ Vng-xanh
Catalase
Khng c bt kh
Xut hin bt kh
SPOR
Bo t
Khng c
GRAM
Hng
Tm
COCC
Que
Cu
Hnh thi
30
VSV trong mi
trng MRS lng
31
2.3.3.2. Tch DNA vi khun bng WizardR Genomic DNA Purification Kit
Nguyn tc:
Tch chit DNA ca vi sinh vt nghin cu: Thnh t bo vi sinh vt c ph
v nh lysozyme v dung dch ly gii t bo gip gii phng ton b DNA ca vi
sinh vt. Protein v RNA c tch khi hn hp nh enzym RNAse v dung mi
kt ta protein. Cui cng DNA c ta bng Isopropanol.
DNA ca cc chng nghin cu v ca cc vi sinh vt phn lp trong cc mu
c tch v tinh sch bng kit ca Promega c ci tin. Quy trnh thc hin nh
sau:
- Cc chng chun c tng sinh t cc ng gc bng mi trng MRS
lng, 370C trong 48 gi. i vi cc mu nghin cu, tip tc tng sinh
VSV trong mi trng MRS lng t phn sinh khi ca VSV c phn lp
sau khi nh danh n chi Bifidobacterium (mc 2.3.2 )
- Ly 1,0 1,5ml mi trng tng sinh chng trn vo ng nha 1,5ml.
- Mang ly tm 10000-12000 vng/pht trong 2 pht loi dch ni thu ly cn.
- Thm 480l dung dch EDTA 50mM vo ng cn, lc u.
- Thm tip 120l dung dch lysozyme (nng 20mg/ml) trn u.
- 370C trong 30-60 pht, sau ly tm 10000-12000 vng/pht trong 2
pht. B dch ni thu ly cn.
- Thm 600l dung dch Nuclei Lysis vo ng cn va thu c. Dng pipet
o nh to thnh hn dch.
- hn dch trn 800C trong 5 pht ly gii t bo, sau ngui n
nhit phng
- Thm 3l dung dch RNAse vo dch ly gii t bo, trn u bng cch o
ng t 5-7 pht.
- 370C trong 15-60 pht, ngui n nhit phng.
- Thm 200l dung dch Protein Precipitation (dung dch kt ta protein)
vo hn hp trn, lc mnh trong 20 giy.
- trong lnh 5 pht.
- em ly tm 10000-12000 vng/pht trong 3 pht.
32
- Chuyn dch ni cha DNA vo mt ng nha 1,5ml sch khc cha 600l
isopropanol. Trn u bng cch o ngc ng t 5-7 ln.
- Ly tm 10000-12000 vng/pht trong 2 pht.
- Loi b dch ni, thu ly cn. Thm 600l dung dch ethanol 70% vo ng,
o nh nhng ra cn DNA.
- Ly tm 10000-12000 vng/pht trong 2 pht. Cn thn loi ht ethanol
tha, kh t nhin trong 10-15 pht.
- Thm 100l dung dch DNA Rehydration (dung dch ha tan DNA) vo
ng cha cn DNA, 650C trong 1 gi.
- Bo qun dung dch DNA va tch c -200C.
33
Loi
Trnh t
Kch
Ti
thc
liu
gen ch
tham
kho
on
ID16R08
16S
rDNA
IDL16R0
5-GGGGGATCCAGAGTTTGATCCTGGCTCA-3
5-GGGCTCGAGTACCTTGTTACGACTTCACC-3
1500 bp
[20]
831 bp
[27],
9R
B.
BlonF
longum
BlonR
[22]
Vector
T7-Fw
5 AATACGACTCACTATAG 3
pGemT
nguyn
bn
[23]
SP6-Rv
5 TATTTAGGTGACACTATAG 3
186 bp
34
Bng 2.6. Thnh phn ca phn ng PCR kim tra DNA mi tch
STT
Thnh phn
Th tch (l)
m PCR (10X)
2,5
dNTPs (2mM)
2,5
0,75
0,1
Nc ct
14,15
25l
Chu trnh nhit cho phn ng PCR nhn bn on gen 16S rDNA: 940C trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 940C
trong 45 giy lm bin tnh DNA, 560C trong 45 giy gn mi v 720C trong 1
pht 30 giy ko di chui. Mu sau c tip 720C trong 5 pht v gi
40C cho n khi phn tch.
* in di trn gel agarose
Chun b bn gel c nng 1% (kl/tt) trong m TBE (pH 8,0) (Tris-Boric
acid-EDTA), nhum sn phm theo k thut in di trn gel gii thiu mc
1.3.5.2 b, km theo mt ging nh cha thang kch thc DNA chun. in di bng
my in di gel XL-ULTRA-V-2 100V trong 30 pht. Nhum sn phm bng
dung dch ethidium bromide 0,1% trong 15-30 pht. Ghi li kt qu bng my chp
nh bn gel UVDI- 312.
35
Thnh phn
Th tch (l)
m PCR (10X)
2,5
dNTPs (2mM)
2,5
0,75
BlonF (10pmol)
BlonR (10pmol)
0,1
Nc ct
14,15
25l
Chu trnh nhit cho phn ng PCR nhn bn on gen c hiu: : 94oC trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 940C
trong 45 giy lm bin tnh DNA, 56oC trong 45 giy gn mi v 720C trong
30 giy ko di chui. Mu sau c tip 72oC trong 5 pht v gi 4oC
cho n khi phn tch.
36
37
Thnh phn
Th tch (l)
m phn ng 2X
10 l
Sn phm PCR
3 l
Vector u T pGEMT-T
1 l
1 l
5 l
Tng th tch
20 L
l, nng
38
39
K hiu
Lactobacillus rhamnosus
ATCC9595
Lactobacillus acidophilus
ATCC 4356
Lactobacillus plantarum
ATCC 8014
Lactobacillus casei
ATCC 334
Lactobacillus fermentum
ATCC 9338
Streptococcus faecalis
ATCC 33186
Bifidobacterium longum
JCM 1217
Streptococcus thermophilus
ATCC 19258
STT
40
xi
S ri
i 1
n 1
Trong :
- xi: Kt qu ca mi ln lp li
- x : Kt qu trung bnh ca cc ln lp li
- n: S ln lp li (n=10)
2.3.4.3. Thm nh qui trnh nh danh bng k thut PCR v gii trnh t
a. nh gi c hiu
41
Nguyn tc: Trong cng mt phn ng PCR tin hnh nhn bn on gen c
hiu ca i tng nghin cu vi s c mt DNA ca nhiu loi khc.
Bng 2.10. Cng thc tnh c hiu
Chng chun
Php th
K thut PCR
Cng
Dng tnh
m tnh
Dng tnh
a+b
m tnh
c+d
a+c
b+d
a+b+c+d
Cng
42
43
nh gi kt qu
Khng c bng
(-)
(+)
(++)
(+++)
44
CHNG 3. KT QU NGHIN CU
3.1. XC NH S LNG VI SINH VT TRONG CH PHM
PROBIOTICS
nh lng vi sinh vt trong cc mu A, B, C, D, E bng phng php ghi
mc 2.3.1. Kt qu c ghi trong Bng 3.1.
Bng 3.1. S lng vi sinh vt trong ch phm
Kt qu
Stt
MU
(CFU/gi)
1, 17
108CFU/gi
108 CFU/gi
2,83 x 108CFU/gi
3,22 x 108CFU/gi
45
46
47
TT
Mu A
Mu B
Mu C
Mu D
Mu E
Ging
Cht nn
IND
L-tryptophane
URE
Urea
GLU
D-glucose
MAN
D-mannitol
LAC
D-lactose
SAC
D-saccharose
MAL
D-maltose
48
SAL
Salicin
XYL
D-xylose
10
ARA
L-arabinose
11
GEL
Gelatin
12
ESC
Esculin ferric
citrate
13
GLY
Glycerol
14
CEL
D-cellobiose
15
MNE
D-mannose
16
MLZ
D-melezitose
17
RAF
D-raffinose
18
SOR
D-sorbitol
19
RHA
L-rhamnose
20
TRE
D-trehalose
CAT (catalase)
Bo t
Gram
Hnh cu
49
Tn mu
Kt qu nh danh
tin cy
Phn ng nghi ng
Bifidobacterium spp1
94,3%
RAF 91%
Actinomyces israelii
4,5%
XYL
A
99%,
RAF
Actinomyces israelii
97,1%
XYL
2,2%
Bifidobacterium spp1
97,1%
Actinomyces israelii
2,2%
99%,
RAF
99%,
RAF
82%,
C
XYL
82%,
Bifidobacterium spp1
94,3%
RAF 91%
Actinomyces israelii
4,5%
XYL
D
99%,
RAF
Bifidobacterium spp1
97,1%
Actinomyces israelii
2,2%
XYL
99%,
RAF
82%,
50
hng
Promega
Tin hnh tng sinh chng B. longum chun v cc vi sinh vt trong cc mu
probiotics trong phn i tng nghin cu phn lp c mc 3.1, tin hnh tch
DNA theo hng dn ti mc 2.3.3.2. Tin hnh o quang cc dung dch DNA mi
tch theo m t trong mc 2.3.3.3 v pha long cc dung dch DNA bng nc ct
siu tinh khit v khun thu c dung dch DNA c nng 50 ng/l.
Bng 3.4. Kt qu o quang cc dung dch DNA mi tch
i tng
Atn
B. longum
0,1125
562,5
Mu A
0,0911
455,5
Mu B
0,0956
478
Mu C
0,1174
587
Mu D
0,0851
425,5
Mu E
0,1052
526
51
1500bp
bp
T Hnh 3.7 cho thy, phn ng PCR nhn bn on gen 16S rDNA c hiu
u cho cc bng nhn bn kch thc 1500 bp, trong khi mu chng m (khng
c DNA) khng xut hin bng nhn bn no. Kt qu ny cho thy thu c cc
khun DNA ca cc i tng nghin cu. DNA sau khi tch chit c bo qun
-200C dng cho cc th nghim tip theo.
52
Thnh phn
Th tch ( l)
m PCR(10X)
2,5
dNTPs (2mM)
2,5
0,75
0,1
(5u/ l)
Nc ct
14,15
25
Vi thnh phn nh trn tin hnh phn ng PCR vi chu trnh nhit:
94oC 45 giy
94oC 5 pht
56oC 45 giy
x 30 chu k
72oC - 5 pht
4oC
72oC 30 giy
Kt qu phn ng PCR c in di trn gel agarose 1% v c ghi li kt
qu bng my chp nh bn gel UVDI-312 (Hnh 3.8)
53
M 1 2
6 7
800 bp
54
831 bp
55
56
57
M 1 2 3 4 5 6 7
M 8 9 10 11 12 13 14
1017bp
831 bp
58
khng thu c bng DNA no (ging 2, 9). Kt qu ny cho php chng ti khng
nh plasmid tinh sch c l plasmid ti t hp mang cc on gen phn lp c
t genome ca loi B. longum
vi enzyme ct gii hn
khng nh chc chn on DNA c chn vo vector nhn dng l
on gen c kch thc 831bp, chng ti tin hnh th nghim ct gii hn plasmid
ti t hp c tinh sch t khun lc dng tnh. Theo l thuyt, trn vector
pGEMT Easy (Hnh 3.10) c hai v tr ct gii hn ca EcoRI nm trong vng a
im ct. Nh vy, khi c x l bng EcoRI vector ti t hp s b ct thnh 2
on DNA, mt on chnh l cc on gen c chn vo v mt on l b khung
vector c kch thc 3 kb.
M1 M2 1 2 M2 3 4 M2 5 6 M2 M2 7 8 M2 9 10 M2 11 12 M1
3000
KL xanh
831
831
831
831
831
3000
59
60
TGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAA
AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCG
TATACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGT
CGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAA
GGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAGCGGGCGGA
GCATGCGGAATTATTCGATGCAACGCGAAGAACCCTTACCCTGGGG
CTTGACATGTTTCCCGGACGGTTGGTTAGAAGAATAACGGGC
Kt qu gii trnh t trn c chng ti tin hnh so snh trnh t vi cc
trnh t khc trn ngn hng gen th gii bng ng dng BLAST (Basic Local
Alignmen CSearch Tool) trn trang web ca NCBI (National Center for
Biotechnology Information-http://blast.ncbi.nlm.nih.gov/Blast.cgi) [37].
Kt qu blast khng nh on gen nhn bn c chnh l on gen c
phn lp t h gen ca Bifidobacterium longum.
61
Tn chng
10-5
10-6
10-7
10-5
10-6
10-7
Lactobacillus
Khng
Khng
Khng
Nhiu
45 +37
4 +3
mc
mc
mc
Lactobacillus ATCC
Khng
Khng
Khng
Nhiu
40+ 50
2+4
4356
mc
mc
mc
Khng
Khng
Nhiu
35+29
2+2
Nhiu
48 +39
3+5
Nhiu
37+ 46
3+3
Nhiu
50 +57
5+4
Nhiu
48+ 38
4+4
mc
mc
mc
Lactobacillus casei
Khng
Khng
Khng
ATCC 334
mc
mc
mc
Lactobacillus
Khng
Khng
Khng
mc
mc
mc
Streptococcus faecalis
Khng
Khng
Khng
ATCC 33186
mc
mc
mc
45 +34
3 +4
62
Hnh 3.14. Hnh thi khun lc B. longum trn mi trng BSM agar
63
Nng 10-6
a 1 a 2 a 3 TB CFU/gi
Ln 1
30
36
27
31
96x106
Ln 2
29
40
42
37
114x106
Ln 3
34
38
47
40
124x106
Ln 4
30
35
42
36
111x106
Ln 5
41
31
31
34
105x106
Ln 6
48
33
36
39
120x106
Ln 7
40
30
36
35
108x106
Ln 8
29
39
31
33
102x106
Ln 9
31
35
43
36
111x106
Ln 10
37
30
28
32
99x106
64
Hnh 3.15. nh danh B. longum JCM 1217 bng kit Api 20A
Kt qu nh danh cho thy vi sinh vt thuc chi Bifidobacterium vi % ID l
97,1%
Nh vy phng php nh tnh vi sinh vt n chi Bifidobacterium bng kit
Api 20A c c hiu cao.
65
Tn chng
Kt qu
TT
Tn chng
Kt
qu
PCR
PCR
1
11
11778
2
Bacillus
ATCC 9338
-
12
14884
3
Lactobacillus fermentum
Lactobacillus plantarum
ATCC 8014
-
13
stearothermophilis
Bifidobacterium breve
ATCC 15700
ATCC 795
4
14
6633
5
Lactobacillus rhamnosus
Lactobacillus casei
15
Lactobacillus
16
Bacillus coagulans
17
Bifidobacterium longum
18
Lactobacillus
Pseudomonas
Escherichia coli
ATCC8739
+
19
JCM 1217
10
Streptococcus pyogenes
ATCC 7050
9
ATCC 19615
Streptococcus faecalis
ATCC 33186
ATCC 334
7
ATCC11863
ATCC 9595
6
Bifidobacterium bifidum
Staphylococcus aureus
ATCC6538
-
20
Salmonella typhimurium
ATCC 14028
66
Ghi ch: (-): Kt qu PCR m tnh: khng xut hin bng nhn bn.
(+): Kt qu PCR dng tnh: xut hin bng nhn bn
M
1 2 3 4 5
6 7 8
9 10 11 12 13
M 14 15 16 17 18 19 20
831bp
Php th
K thut PCR
Cng
Dng tnh
m tnh
Dng tnh
a (1)
b (0)
a+b
m tnh
c (0)
d (19)
c+d
a+c
b+d
a+b+c+d
Cng
67
10-7
10-8
a 1(CFU/a)
Nhiu
115
a 2 (CFU/a)
Nhiu
127
pha long
Trung bnh
121
1,21 x 109
Dd D (CFU/ml)
68
Trong dch
chit DNA
tch chit
(copy/l)(*)
Kt qu PCR trong cc ln th
1
(CFU/ml)
109
107
++
++
++
108
106
++
++
++
107
105
++
++
++
106
104
++
++
105
103
104
102
(*) V DNA sau khi tch chit c ha tr li trong 100l DNA Rehydration
Solution, nn s lng bn copy ca phn t DNA trong dung dch sau khi tch
chit s gim 100 ln so vi s lng vi khun bt u trong hn dch trc khi
tch chit.
69
Kt qu cho thy:
- Ngng pht hin ca quy trnh nhn bn on gen c hiu cho B. longum
l 105 CFU/ml vi khun (tng ng vi khong 103copy/l dng lm khun
cho phn ng PCR). Kt qu c th hin nh Hnh 3.17
3 4
831 bp
Ging 1-6: Sn phm PCR vi khun l DNA tch chit t nng chng t
109 CFU/ml vi khun n 104 CFU/ml vi khun (tng ng vi khong 107
copy/ l n 102 copy/ l dng lm khun cho phn ng PCR)
70
CHNG 4. BN LUN
Hin nay, cc ch phm probiotics lu hnh trn th trng hu ht l cc sn
phm hn hp ca nhiu loi vi khun khc nhau. Tuy nhin cc tiu chun c s
do cc nh sn xut a ra cn rt nhiu thiu st. Vic nh lng ch dng li
vic m tng s vi sinh vt trong mu. Trong a s cc trng hp, nh danh
cn rt s si, ch dng li phn ng nhum soi v mt vi phn ng sinh ha.
Do , nu thc hin theo tiu chun, rt kh kt lun mu t hay khng v s
nh hng n quyn li ca bnh nhn. Xut pht t thc t, nhu cu cp thit
t ra l phi nghin cu nh lng, nh danh n loi i vi cc vi sinh vt
c ghi trn nhn ca ch phm. Cng vi s pht trin ca khoa hc k thut,
vic nh danh vi sinh vt khng ch bao gm kiu hnh m cn phi nh danh
kiu gen.
Nghin cu trc y ca Nguyn Th Thu Hng [4] ng dng k thut
PCR nh danh mt s vi sinh vt thuc chi Lactobacillus trong hn hp nhiu
vi sinh vt bng cch tch chit DNA trc tip t mu. Trong nghin cu ny, tc
gi thnh cng trong vic nh danh n loi mt s Lactobacillus trong hn
hp gm nhiu vi sinh vt thuc chi Lactobaccillus m bng cc phn ng sinh
ho khng th thc hin c. Tuy nhin, nhc im ca k thut ny l khng
phn bit c vi khun sng hay vi khun cht. Vi quy trnh xy dng c
trong ti ny, chng ti nh lng ring c B. longum trong hn hp
ng thi phn lp c khun lc thun khit tin hnh nh danh kiu hnh
bng kit sinh ha v nh danh kiu gen bng k thut PCR v gii trnh t. Do
s khng nh c chc chn s lng B. longum c mt trong hn hp nng
l bao nhiu. iu ny rt c ngha trong vic kim tra gim st cht lng
thuc.
ng dng quy trnh thit lp, chng ti kim tra c mt s mu lu
hnh trn th trng, khng nh c chc chn cc sn phm ny u c cha B.
longum nh trn nhn do nh sn xut cng b. Vi nhng mu probiotics m nh
sn xut ch cng b tng s lng vi sinh vt trong hn hp (L-Bio-3D, Lackid,
71
72
73
TING ANH
9. Akatsu H, Iwabuchi N, Xiao JZ, Matsuyama Z, Kurihara R, Okuda K,
Yamamoto T, Maruyama M (2012), Clinical Effects of Probiotic
Bifidobacterium longum BB536 on Immune Function and Intestinal
Microbiota in Elderly Patients Receiving Enteral Tube Feeding, JPEN J
Parenter Enteral Nutr, 37(5), p. 631-640.
74
75
20. Kwon HS, Yang EH, Yeon SW, Kang BH, Kim TY (2004), Rapid
identification of probiotic Lactobacillus species by multiplex PCR using
species-specific primers based on the region extending from 16S rRNA
through 23S rRNA., FEMS Microbiology Letters, 239, p 267-275.
21. Lahtinen SJ, Gueimonde M, Ouwehand AC (2006), Comparison of four
methods to enumerate probiotic bifidobacteria in a fermented food product,
Food microbiology, 23, p. 571-577.
22. Matsuki T, Watanabe K, Tanaka R, Fukuda M and Oyaizu H (1999),
Distribution of Bifidobacterial Species in Human Intestinal Microflora
Examined with 16S rRNA-Gene-Tareted Species Specifics Primers,
Applied and Enviroment Microbiology, 65 (10), p. 4506-4512.
23. Promega Technical manual, pGEM-T and pGEM-T easy vector system, USA,
6/2009.
24. Schell
MA, Karmirantzou
M, Snel
B, Vilanova
D, Berger
B, Pessi
76
77
PH LC
Ph lc 1.
Ph lc 2.
Ph lc 3.
Ph lc 4.
Ph lc 5.
Ph lc 6.
Ph lc 7.
Ph lc 8.
Ph lc 9.
Ph lc 10.
Ph lc 11.
Ph lc 1
Ph lc 2
Ph lc 3
Ph lc 4
Ph lc 5
Ph lc 6
Ph lc 7
Kt qu gii trnh t ca VSV phn lp t mu SamubioTHYMO
CGGTTTGCATCCAACGCGTTGGTGAGCTCTCCCATATGGTCGACCTGCA
GGCGGCCGCGAATTCACTAGTGATTTTCCAGTTGATCGCATGGTCTTCTG
GGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCCGCTTGACGG
CGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGG
CGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGG
CAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACG
CCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGA
GCAAGCGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTG
CCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTG
GGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCAT
CGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAG
GGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGA
AGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAG
GAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC
GCCGTAGACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTG
TCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAGGGC
TAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGC
GGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTC
CCGACGGTCGTAGAGATACGGCTTCCCAATCGAATTCCCGCGGCCGCCA
TGGCGGCCGGGAGCATGCGACGTCAGGCCCAATTCGCCCTATATGGAGG
TCGTATTAAT
Ph lc 8
Kt qu gii trnh t ca VSV phn lp t mu Safikid BIO
CGCGGGTCTATGTGATCTTACAGTGCTCCCGGCCGCCATGGCGGCCGCG
GGAATTCGTATCTGGGAAGCCGTATCTCTACGACCGTCGGGAACATGTC
AAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCC
GCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCC
GTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACACGGAACCCG
TGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCA
GGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTA
ACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACAC
ATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCC
GCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGG
ACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAAC
GCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTG
CTTATTCAACGGGTAAACTCACTCTCGCTTGCTCCCCGATAAAAGAGGTT
TACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTT
GCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGC
CGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGT
CGAAGCCACGGTGGGCCGTTACCCCGCCGTCAAGCGGATAGGACGCGA
CCCCATCCCATACCGCGAAAGCTTTCCCAGAAGACCATGCGATCAACTG
GAAAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGAG
AGCTCCCAACGCGTTGGATGCATAGCTTGAGTATCTATAGTAGTCACCT
AAAT
Ph lc 9
Kt qu gii trnh t ca VSV phn lp t mu Lackid
TGGGTGGGTACACGCATTGGGAGGGTCCCACATGGCGACCTGCAGGCGT
CCGCGAATTCACAAATGATTTTCCAGTTGATCTTATGGTCTTCTGGGAAA
GCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCCGCTTGACGGCGGGG
TAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACC
GGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCA
GTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCG
TGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAG
CGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGC
AGCCGCGGTAATACGTAGGGTGGAAGCGTTATCCGGAATTATTGGGCGT
AAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTT
AACGGTGGATCCGCGCCGGGTAAGGGCGGGCTTGAGTGCGGTAGGGGA
GACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAAC
ACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCG
AAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGT
AAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGA
GCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCGAGGCTAAA
ACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGAT
TAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGA
CGGTCGTAGAGATACGGCTTCCCAATCGAATTCCCGCGACCGCCATGGC
GGCCGGGAGCATGCGACGTCGGGCCCATTTCGCCCTATAGTGAGTCGTA
TTACAATTCACTGGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCC
TGGCGTTACCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG
GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCA
GCCTGAATGGCGAATGGACGCGGCCTGTAGCGGCGCATTAAGCGCGGC
GGGGGGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCAGGGCCTAG
CGCCGCTCCTTTCCCTTTCTTCCCTTCTTTCTCGCCCGTTCGCGGGTTCCC
GTCAAGTTTAATCGGGGGCTCCCTTTAGGTTCGATTATGGCTTTACGGAC
CTCAACCCCAAAACTTGATAAGGGGGTGGTCAGGGTTTG
Ph lc 10
Kt qu gii trnh t ca VSV phn lp t mu Probiotics Lactomin Plus
CCGATGTTCTCTGTACTCTCGTCGAGTCGTTCTCTGCGCTCCGGCCGCCT
GGCGGCCGCGGGAATTCGTTCTGGGAAGCCGTATCTCTACGACCGTCGG
GAATTGCCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCG
CATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCC
TTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACAC
GGAACCCGTGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTG
GACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG
CGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGAT
ATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCAC
TCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTT
CACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCGAATAATTC
CGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTG
AGCCGGTGCTTATTCAACGGGTAAACTCACTCTCGCTTGCTCCCCGATAA
AAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGGTCGCTGCTC
AGGCTTGCGCCCATTGTACAATATTCCCCACTGCTGCCTCCCGTAGGATC
TGGGCCGTATCTCATCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGTAC
CCGTCAAAGCCCGGGGGGGCCGTTACCCCGCCGTCAAGCGGATAGGAC
GCGACCCCATCCATACCGGGAAAGCTTTCCCAGAAGACCATGCGTTCAA
CTGGAAAAATCACTAATGAATTCGGGGGCCGCCTGTAGGCGACCATATG
GGAA
Kt qu so snh trnh t gii vi cc trnh t trong ngn hng gen th gii bng
ng dng BLAST
Ph lc 11
1. nh lng B. longum trong cc ch phm probiotics
Cn v xc nh khi lng trung bnh ca 20 gi ch phm. Cn chnh xc
khong 10,0g bt thuc vo bnh nn v trng, thm dung mi pha long vi th
tch tnh theo mililit bng 9 ln khi lng bt thuc cn v vi vin bi thu
tinh v trng. Lc mnh trong 30 pht vi sinh vt phn tn u trong dung dch,
thu c hn dch c pha long 10-1. Ly 1000 l hn dch trn cho vo ng
nghim cha sn 9,0ml dung mi pha long, lc u bng my lc trn Vortex
c hn dch c pha long 10-2. Tip tc pha long 10 ln nh trn thu
c hn dch c c pha long 10-5, 10-6 v 10-7.
Tin hnh: Chun b ba a petri v trng, chuyn vo mi a chnh xc
1000 l hn dch c pha long 10-5. Tip tc lm nh trn vi hn dch c pha
long 10-6 v 10-7. Cy trn vi mi trng BSM c ( c tit trng v ngui
xung khong 45oC), mi trng trong a ng t nhin. Lt ngc a petri v
1oC trong 48 - 72 gi trong iu kin k kh. Sau thi gian , m s lng
37
MTB
Trong :
A
nTB
MTB
i vi ging IND: Thm 1 git thuc th XYL vo lp du khong pha trn, trn
u v 2-3 pht. Sau thm 1 git thuc th EHR. c kt qu trong vng 5
pht. Kt qu l dng tnh nu xut hin mu .
Phn ng CAT: kit tip xc vi khng kh khong 30 pht, sau thm 2 git
thuc th H2O2 3% vo mt ging dng tnh bt k. Kt qu c ghi l dng
tnh nu thy xut hin bt kh.
Cch c ging dng tnh v ging m tnh c trnh by Bng 1
Hm
Ging
Cht nn
lng(mg/gi
Kt qu
Phn ng
ng)
Dng tnh
m tnh
L-tryptophan
0,98
Vng
URE
Urea
0,648
Urease
Vng-cam
BCP
GLU
D-glucose
1,96
Acid ha
MAN
D-manitol
1,96
Acid ha
LAC
D-lactose
1,96
Acid ha
SAC
D-saccharose
1,86
Acid ha
MAL
D-maltose
1,96
Acid ha
SAL
Salicin
1,64
Acid ha
XYL
D-xylose
1,64
Acid ha
ARA
L-arabinose
1,64
Acid ha
GEL
gelatin
0,6
ESC
Esculin
0,36
St (III) citrat
0,11
Tm
Vng/ vng-xanh
C s khuch tn
mu
cht mu en
Vng
Nu-en
Soi di n UV 365nm
S thy phn Esculin
Pht quang
GLY
Glycerol
1,82
Acid ha
CEL
D-cellobiose
1,86
Acid ha
MNE
D-mannose
1,96
Acid ha
MLZ
D-melezitose
1,96
Acid ha
RAF
D-raffinose
2,18
Acid ha
SOR
D-sorbitol
2,18
Acid ha
RHA
L-rhamnose
1,96
Acid ha
TRE
D-trehalose
1,96
Acid ha
Tm
Vng/ Vng-xanh
Catalase
Khng c bt kh
Xut hin bt kh
SPOR
Bo t
Khng c
GRAM
Hng
Tm
COCC
Que
Cu
Hnh thi
VSV trong mi
trng MRS lng
3.2. Tch DNA vi khun bng WizardR Genomic DNA Purification Kit
DNA ca cc chng nghin cu v ca cc vi sinh vt phn lp trong cc
mu c tch v tinh sch bng kit ca Promega c ci tin. Quy trnh thc hin
nh sau:
Cc chng chun c tng sinh t cc ng gc bng mi trng MRS lng,
370C trong 48 gi. i vi cc mu nghin cu, tip tc tng sinh VSV trong mi
trng MRS lng t phn sinh khi ca VSV c phn lp sau khi nh danh
n chi Bifidobacterium (mc 2 )
-
Ly 1,0 1,5ml mi trng tng sinh chng trn vo ng nha 1,5ml. Mang ly
tm 10000-12000 vng/pht trong 2 pht loi dch ni thu ly cn.
Thm 3l dung dch RNAse vo dch ly gii t bo, trn u bng cch o ng t
5-7 pht. 370C trong 15-60 pht, ngui n nhit phng.
Chuyn dch ni cha DNA vo mt ng nha 1,5ml sch khc cha 600l
isopropanol. Trn u bng cch o ngc ng t 5-7 ln. Ly tm 10000-12000
vng/pht trong 2 pht. Loi b dch ni, thu ly cn.
Thm 100l dung dch DNA Rehydration (dung dch ha tan DNA) vo ng cha
cn DNA, 650C trong 1 gi.
Thnh phn
Th tch (l)
m PCR (10X)
2,5
dNTPs (2mM)
2,5
0,75
0,1
Nc ct
14,15
25l
Chu trnh nhit cho phn ng PCR nhn bn on gen 16S rDNA: 940C trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 940C
trong 45 giy lm bin tnh DNA, 560C trong 45 giy gn mi v 720C trong 1
pht 30 giy ko di chui. Mu sau c tip 720C trong 5 pht v gi
40C cho n khi phn tch.
* in di trn gel agarose
Thnh phn
Th tch (l)
m PCR (10X)
2,5
dNTPs (2mM)
2,5
0,75
BlonF (10pmol)
BlonR (10pmol)
0,1
Nc ct
14,15
25l
Chu trnh nhit cho phn ng PCR nhn bn on gen c hiu: : 94oC trong
5 pht bin tnh hon ton DNA khun, tip theo l 30 chu k ca 3 bc: 94 0C
trong 45 giy lm bin tnh DNA, 56oC trong 45 giy gn mi v 720C trong
30 giy ko di chui. Mu sau c tip 72oC trong 5 pht v gi 4oC
cho n khi phn tch.
tch bng khi lng ming gel (1l : 1 mg) v trong b n nhit 60C trong
khong 10 pht n khi gel tan hon ton.
-
Thnh phn
Th tch (l)
m phn ng 2X
10 l
Sn phm PCR
3 l
Vector u T pGEMT-T
1 l
1 l
5 l
Tng th tch
20 L
l, nng
Ct tinh sch cha plasmid c chuyn sang ng eppendorf 1,5 ml mi. B sung
50 l m y (Elution buffer) vo chnh gia mng silica ca ct v nhit
phng 2 pht.