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PHM TH NHN
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
NI DUNG BO CO
1 2 3 4
M u
Kt qu v tho lun
MC TIU NGHIN CU
Thit k cc phn ng Multiplex PCR xc nh
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.
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)
AF532128.1 AF532129.1
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
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
2,5
1,3 2,5 0,4 0,4 0,4 0,4 0,4 0,4 3 0,5
7 8
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
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
872 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
EPEC EHEC1 + + + -
EHEC2 + + -
EAEC + -
EIEC + -
ETEC +
EC -
620 bp 376 bp
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
Chng
EHEC2 EHEC1
ETEC EPEC EIEC
2 1
1 1 1
1 bng, 1 thp hn
Cao hn Bng
Cao hn Cao hn Cao hn
1 2
2 1 1
Thp hn
Thp hn Bng Cao hn
EAEC
Cao hn
Cao hn
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
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
Mu
11 12 13 14 15 16 17 18 19
Tui
3
a ch
ng H - TN
Chn on
m tnh
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
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
Multiplex PCR 1
Multiplex PCR 2
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