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vn
PHM TH NHN

I HC THI NGUYN TRNG I HC KHOA HC ------------------

NG DNG K THUT MULTIPLEX PCR NHM CHN ON CC CHNG E.COLI GY BNH TIU CHY NGI
KHA LUN TT NGHIP I HC
Ngi hng dn khoa hc: ThS. NGUYN PH HNG

THI NGUYN - 2010

Ti liu chia s ti: wWw.SinhHoc.edu.vn

NI DUNG BO CO

1 2 3 4

M u

i tng v phng php nghin cu

Kt qu v tho lun

Kt lun v kin ngh

Ti liu chia s MwWw.SinhHoc.edu.vn ti: U


Tiu chy l mt vn sc khe ton cu. Mt trong nhng nguyn nhn ph bin gy tiu chy tr em l do vi khun E.coli. Vic xc nh nhanh cc nhm E.coli bng cc k thut c nhy v c hiu cao s c ngha rt quan trng trong vic la chn phng php iu tr thch hp cho bnh nhn. C nhiu phng php c s dng xc nh E.coli gy tiu chy. Trong , Multiplex PCR l phng php c s dng hu hiu nht.

Xut pht t nhng l do trn, chng ti tin hnh thc hin


ti ng dng k thut Multiplex PCR nhm chn on cc chng E.coli gy bnh tiu chy ngi.

MC TIU NGHIN CU
Thit k cc phn ng Multiplex PCR xc nh

nhanh cc chng E.coli, lm c s to b Kt chn


on nhanh cc tc nhn E.coli gy tiu chy ngi. p dng k thut Multiplex PCR chn on nhanh tc nhn E.coli gy tiu chy trn cc mu bnh phm Xy dng quy trnh chn on cc tc nhn E.coli gy tiu chy p dng mc phng th nghim.

NI DUNG NGHIN CU
Tch DNA tng s t khun lc E.coli.

Khuch i cc gen c lc ca 6 chng E.coli bng phn ng PCR. Khuch i cc gen c lc ca 6 chng E.coli bng phn

ng Multiplex PCR.
ng dng k thut Multiplex PCR chn on nhanh cc tc nhn E.coli gy tiu chy trn 20 mu bnh phm. Xy dng quy trnh chn on cc tc nhn E.coli gy tiu chy p dng mc phng th nghim.

Ti liu chia s PHNG PHP NGHIN CU I TNG V ti: wWw.SinhHoc.edu.vn


1. I TNG NGHIN CU
7 chng E.coli chun quc t do Phng Vi khun

ng rut - Vin V sinh Dch t Trung ng cung

cp, gm c: EPEC, EHEC1, EHEC2, ETEC, EIEC,


EAEC, EC.
20 mu bnh phm thu nhn ti Phng xt nghim

Vi sinh - Bnh vin a khoa Trung ng Thi Nguyn.

2.Ti liu chia s ti: wWw.SinhHoc.edu.vn PHNG PHP NGHIN CU


Cc chng E.coli chun Quc t Mu bnh phm nghi l E.coli ly ti bnh vin

Nui cy qua m trn mi trng MPA c, thu khun lc

Tch DNA trc tip t khun lc

PCR

Multiplex PCR

in di kim tra sn phm trn gel agarose Hnh 2.1. S cc bc c thc hin trong qu trnh nghin cu

Bng 2.1. Cc trnh s mi c s dng trong nghin cu Ti liu chia t ti: wWw.SinhHoc.edu.vn
Gen M s trn GenBank Cp mi
VT2 F VT2 - R VT1 F VT1 - R LT F LT - R FJ666096.1 eae F eae - R IpaH F IpaH - R EA F EA - R

Trnh t primer (5 3)

on DNA khuch i (bp)


298 130 322 376 620 630

vt2 vt1 elt eae IpaH


EAST1

AF532128.1 AF532129.1

ACCGTTTTTCAGATTTTGCACATA TACACAGGAGCAGTTTCAGACAGT GAAGAGTCCGTGGGATTACG AGCGATGCAGCTATTAATAA TCTCTATGTGCATACGGAGC CCATACTGATTGCCGCAAT CACACGAATAAACTGACTAAAATG AAAAACGCTGACCCGCACCTAAAT GTTCCTTGACCGCCTTTCCGATACCG


GCCGGTCAGCCACCCTCTGAGAGTAC

CTGGCGAAAGACTGTATCAT CAATGTATAGAAATCCGCTGTT

Cc cp mi ca cc gen elt, IpaH v EAST1 c k tha t cc nghin cu ca Schmidt H., Svenungsson B. v Sethabutr O.

Ti liu 2.2. Thnh phn phn ng PCR Bng chia s ti: wWw.SinhHoc.edu.vn
STT Thnh phn Th tch (l)

1
2 3 4

Nc kh ion
PCR Buffer (10X) dNTPs (25mM) MgCl2 (25mM)

12,5
2,5 2 2,5

5
6 7 8

Mi xui (10pM/mi)
Mi ngc (10pM/mi) DNA khun Taq DNA polymerase (5 unit/l) Tng th tch

1
1 3 0,5 25

Ti liu chia s ti: wWw.SinhHoc.edu.vn


Bng 2.3. Chu trnh nhit ca phn ng PCR
Nhit 940C 940C 520C 720C 720C 40C Thi gian 3 pht 1 pht 1 pht 1 pht 10 pht S chu k 1 Tc dng Bin tnh Bin tnh Gn mi Tng hp chui Tng hp chui n nh mu Cc bc 1

30

3 4

Ti liu Thnh ti: phn ng Multiplex PCR Bng 2.4. chia sphn wWw.SinhHoc.edu.vn 1
STT
1

Thnh phn
Nc kh ion

Th tch (l)
12,8

2
3 4 5

PCR Buffer (10X)


dNTPs (25mM) MgCl2 (25mM) Mi: VT2 - F VT2 - R eae - F eae - R IpaH - F IpaH - R

2,5
1,3 2,5 0,4 0,4 0,4 0,4 0,4 0,4 3 0,5

7 8

DNA khun Taq DNA polymerase (5 unit/l)

Tng th tch

25

Bng 2.5. chia sphn wWw.SinhHoc.edu.vn 2 Ti liu Thnh ti: phn ng Multiplex PCR
STT
1 2 3 4 5

Thnh phn
Nc kh ion PCR Buffer (10X) dNTPs (25mM) MgCl2 (25mM) Mi: VT1 - F VT1 - R LT - F LT - R EA - F EA - R

Th tch (l)
12,8 2,5 1,3 2,5 0,4 0,4 0,4 0,4 0,4 0,4

7
8

DNA khun
Taq DNA polymerase (5 unit/l) Tng th tch

3
0,5 25

Ti liu chia s ti: ca phn ng Multiplex PCR Bng 2.6. Chu trnh nhit wWw.SinhHoc.edu.vn
Cc bc 1 Nhit 940C 940C 520C 720C 720C 40C Thi gian 3 pht 1 pht 1 pht 1 pht 10 pht S chu k 1 Tc dng Bin tnh Bin tnh Gn mi Tng hp chui Tng hp chui n nh mu

35

3 4

Ti liu chia s ti: wWw.SinhHoc.edu.vn KT QU V THO LUN


1. KT QU TCH DNA TRC TIP T KHUN LC
DNA c tch chit trc tip t khun lc E.coli bng phng php ca Cebula v cng s (1995) c ci tin cho ph hp vi iu kin th nghim.
u im: - Tng thi gian tch DNA l 25 pht. - Khng phi tiu tn nhiu ha cht. - Lng DNA thu c l rt nh, nn c th s dng trc tip lm khun cho cc phn ng PCR (3 l/1 phn ng) m khng cn tin hnh pha long.

2. KHUCH I CC GEN C LC BNG K THUT PCR


M 1 2 3 4 5 6 7

Ti liu chia s ti: wWw.SinhHoc.edu.vn


M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7

872 bp 603 bp 270 bp

376 bp

130 bp

298 bp

Hnh 3.1. Gen eae M. Marker; 1. EPEC; 2. ETEC; 3. EHEC1; 4. EAEC; 5. EHEC2; 6. EIEC; 7. EC

Hnh 3.2. Gen vt1 M. Marker; 1. EHEC1; 2. EC; 3. EAEC; 4. EPEC; 5. EHEC2; 6. EIEC; 7. ETEC

Hnh 3.3. Gen vt2 M. Marker; 1. EHEC1; 2. EHEC2; 3. EPEC; 4. ETEC; 5. EIEC; 6. EAEC; 7. EC

2. KHUCH I CC GEN C LC BNG K THUT PCR


M 1 2 3 4 5 6 7
M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7

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872 bp

630 bp 322 bp 620 bp

603 bp
270 bp

Hnh 3.4. Gen elt M. Marker; 1. EPEC; 2. EHEC1; 3. EHEC2; 4. ETEC; 5. EAEA; 6. EIEC; 7. EC

Hnh 3.5. Gen IpaH M. Marker; 1. EPEC; 2. EIEC; 3. EHEC1; 4. EHEC2; 5. ETEC; 6. EAEC; 7. EC

Hnh 3.6. Gen EAST1 M. Marker; 1. EPEC; 2. EHEC1; 3. EHEC2; 4. EIEC; 5. ETEC, 6. EAEC; 7. EC

Tm tt kt qu khuch i cc gen c lc bng k thut PCR Bng 3.1. Kt qu khuch i cc gen c lc ca cc chng E.coli bng k thut PCR
Chng Gen

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EPEC EHEC1 + + + -

EHEC2 + + -

EAEC + -

EIEC + -

ETEC +

EC -

eae vt1 vt2 IpaH EAST1 elt

Ghi ch: EC l nhm i chng khng c gen c lc + : Dng tnh - : m tnh

3. KHUCH I CC GEN C LC BNG K THUT MULTIPLEX PCR

Ti liu chia s ti: wWw.SinhHoc.edu.vn


1 2 3 M M 4 5 6

620 bp 376 bp

872 bp 603 bp 270 bp 194 bp

630 bp 322 bp 130 bp

Hnh 3.7. Phn ng Multiplex PCR 1


1. EHEC2; 2. EIEC; 3. EPEC; M. Marker

Hnh 3.8. Phn ng Multiplex PCR 2


M. Marker; 4. EHEC1; 5. ETEC; 6. EAEC

Bng 3.2. So snh ti: wWw.SinhHoc.edu.vn Ti liu chia s s tiu hao ha cht gia phn
ng PCR v Multiplex PCR
PCR Thnh phn
Nc kh ion m dNTPs MgCl2 Mi Taq polymerase DNA khun Tng th tch

Multiplex PCR

1 phn ng (l)
14.5 2.5 2 2.5 2 0.5 3 25

6 phn ng (l)
87 15 12 15 12 3 18 150

1 phn ng (l)
12.8 2.5 1.3 2.5 2.4 0.5 3 25

2 phn ng (l)
25.6 5 2.6 5 4.8 1 6 50

Tit kim (l)


56.3 10 7.3 10 8.4 2 12 100

3.4. CHN ON E.COLI GY TIU CHY TRN CC MU BNH PHM

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Gen c nhn ln (kch thc) S bng DNA

Bng 3.3. D on cc chng E.coli da vo mu i chng


V tr bng DNA so vi i chng
gen eae (376 bp) gen vt1 (130 bp) Nhm phn ng Multiplex PCR

Chng

EHEC2 EHEC1
ETEC EPEC EIEC

vt2 (298 bp), eae (376 bp)

2 1
1 1 1

1 bng, 1 thp hn

Cao hn Bng
Cao hn Cao hn Cao hn

1 2
2 1 1

vt1 (130 bp)


Elt (322 bp) Eae (376 bp) IpaH (620 bp)

Thp hn
Thp hn Bng Cao hn

EAEC

EAST1 (630 bp)

Cao hn

Cao hn

Bng 3.4. Kt qu chn on tc nhn E.coli gy tiu chy ca cc bnh nhn

Ti liu chia s ti: wWw.SinhHoc.edu.vn


Tn bnh nhn
Phm Hi Tn Pht Nguyn Hong Dng Nguyn Phan Thi Dng Nguyn Vn Tng Lm Nguyn Thu Phng Lng Vn Chung Trn Th Minh

Mu
1 2 3 4 5 6 7

Tui
2 12 4

a ch
Quyt Thng TN Qun Triu TN Quang Trung TN

Chn on
EHEC2 EIEC EAEC EHEC2 EIEC m tnh EIEC

4 thng Quang Trung TN 11 thng Phan nh Phng TN 52 22 ng H TN ng H TN

8
9 10

L Th Thy
Phm Vn Bc Ng Hong Anh

25
32 3

ng H TN
Ph Lng TN V Nhai - TN

EIEC
m tnh m tnh

Bng 3.4. Kt qu chn on tc nhn E.coli gy tiu chy ca cc bnh nhn

Ti liu chia s ti: wWw.SinhHoc.edu.vn


Tn bnh nhn
o Vn Hi L Anh Trn Th Thanh Tho Nguyn Th Hng Phm Linh an An Th Tuyt Nguyn Khnh Dng L Th Hin Nguyn Th Hng

Mu
11 12 13 14 15 16 17 18 19

Tui
3

a ch
ng H - TN

Chn on
m tnh

17 thng Phan nh Phng TN 4 Quyt Thng TN

m tnh
m tnh

11 thng i T - TN 14 thng Phan nh Phng TN 47 8 thng 9 thng 53 Tn Long TN Phan nh Phng TN Quyt Thng TN Quyt Thng TN

m tnh
m tnh

m tnh
m tnh m tnh m tnh m tnh

20

Mai Th Huyn

Quyt Thng TN

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Hnh 3.9. Hnh nh in di chn on E.coli ca mt s mu bnh phm Ghi ch: C1: EHEC1, chng i chng 1 C2: EPEC, chng i chng 2 1.1; 2.1;; 9.1: Phn ng Multiplex PCR 1 1.2; 2.2;; 9.2: Phn ng Multiplex PCR 2

5. XY DNG QUY TRNH CHN ON MC PHNG TH NGHIM Bnh nhn tiu chy Ly mu bnh phm, nui cy Tch DNA

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Multiplex PCR 1

Multiplex PCR 2

Gen i chng eae

in di kim tra trn gel agarose Phn tch kt qu in di v a ra kt lun chn on

Gen i chng vt1

Hnh 3.10. Quy trnh chn on E.coli gy tiu chy p dng cho cc phng th nghim c trang b thit b PCR

Ti liu chia s ti: wWw.SinhHoc.edu.vn KT LUN V KIN NGH


1. KT LUN Tch chit c DNA tng s ca cc chng vi khun E.coli bng phng php tch DNA trc tip t khun lc, tit kim c nhiu thi gian v ho cht nghin cu.
Khuch i thnh cng cc gen c lc c trng ca 6 chng E.coli bng k thut PCR. Khuch i thnh cng cc gen c lc c trng ca 6 chng E.coli bng k thut Multiplex PCR, tit kim c ti 66,7% lng ha cht tiu hao v gim ng k thi gian chn on so vi PCR n mi. Bc u th nghim thnh cng trong chn on cc tc nhn E.coli gy tiu chy trn 20 mu bnh phm. Xy dng c quy trnh chn on bng k thut Multiplex PCR i vi tc nhn l E.coli gy tiu chy ngi mc phng th nghim.

Ti liu chia s ti: wWw.SinhHoc.edu.vn


2. KIN NGH
ng dng k thut Multiplex PCR trong chn on cc chng E.coli gy tiu chy mc phng th nghim. Tip tc pht trin nghin cu sn xut b Kit Multiplex PCR chn on nhanh cc tc nhn E.coli gy tiu chy ngi.

Xin chn thnh cm n Thy c v cc bn!

TiMT S KT QU LIN QUAN N TI liu chia s ti: wWw.SinhHoc.edu.vn


Gii 3: Gii thng sng to k thut Vit Nam VIFOTEC ln th 18, nm 2010.
Gii 3: Gii thng sinh vin nghin cu khoa hc ton quc ln th 21, nm 2010. 1 bi bo khoa hc: Nguyn Ph Hng, Phm Th Nhn, L Th Thu H, L Thnh Cng, Phng c Cam, Pht hin hai nhm Escherichia coli EPEC v EIEC gy tiu chy ngi bng k thut PCR, Tp ch Khoa hc v Cng ngh, i hc Thi Nguyn, tp 65, s 3, 2010.

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