TRUNG TM QUAN TRC, CNH BO MI TRNG V BNH THY SN VIN NGHIN CU NUI TRNG THY SN I

CHN ON BNH NG VT THY SN BNG PHNG PHP PCR

Bc Ninh, 2006

Mc Lc
Phn 1. Phng php PCR v ng dng PCR .................................................... 2 1. Nguyn tc phn ng PCR.................................................................................. 2 2. Mt s nhn t chnh nh hng n phn ng PCR......................................... 3 3. ng dng ca PCR trong vic chn on bnh ng vt Thy sn .................. 3 Phn II. Trang thit b v ha cht ........................................................................ 4 1. Trang thit b....................................................................................................... 4 2. Ha cht .............................................................................................................. 5 3. Cc loi Kt thng dng.................................................................................... 6 Phn III. Phng php chn on bnh ng vt Thy sn bng phng php PCR .................................................................................................................. 7 1. Quy trnh chn on............................................................................................ 7 2. Phng php chn on bnh MBV trn tm s................................................ 8 3. Chn on tc nhn gy bnh m trng (WSSV) trn tm s v tm Chn trng ......................................................................................................................12 4. Chn on bnh u vng trn tm s bng phng php RT - PCR s dng kt IQ2000YHV/GAV...........................................................................................18 5. Chn on hi chng Taura bng phng php RT- PCR s dng kt IQ2000TSV...........................................................................................................22 5. Chn on bnh VNN trn c bin bng phng php RT- PCR s dng kt IQ2000VNN..........................................................................................................26

Phn 1. Phng php PCR v ng dng PCR

1. Nguyn tc phn ng PCR Phng php PCR (Polymerase Chain Reaction) c Kary Mullis v cng s (M) pht minh nm 1985 v k t to nn mt tc ng to ln i vi cc nghin cu sinh hc trn ton th gii. y l phng php in vitro s dng cc cp mi tng hp s lng ln cc bn sao t mt trnh t ADN c bit da trn hot ng ca enzyme polymerase. Phng php PCR da trn hot ng ca ADN polymerase trong qu trnh tng hp ADN mi t mch khun. Tt c cc ADN polymerase u cn nhng mi, l nhng on ADN ngn c kh nng bt cp b sung vi mt u ca mch khun. on mi ny sau s c ni di ra nh hot ng ca ADN polymerase hnh thnh mt mch mi hon chnh. Mt phn ng PCR l mt chui nhiu chu k ni tip nhau, mi chu k gm ba giai on: - Giai on bin tnh: tch chui ADN t mch i thnh dng mch n - Giai on bt cp: gn cp mi c trng theo nguyn tc b sung - Giai on ko di chui: tng hp chui AND mi ging chui AND gc .

2. Mt s nhn t chnh nh hng n phn ng PCR Cp Mi Cc mi c chn phi c trng cho trnh t ADN cn khuch i. ADN mu Kt qu phn ng PCR l thuc rt ln vo ADN mu, phn ng PCR ti u khi ADN tht tinh sch. 3. ng dng ca PCR trong vic chn on bnh ng vt Thy sn Hin nay, phng php ny c s dng rng ri trong nhiu nghin cu sinh hc phn t, vi sinh, kim nghim, chn on pht hin mm bnh, vi sinh vt, virus, to t bin gene v xc nh cc mi quan h h hng v di truyn ca cc loi ng vt v thc vt.trong Thy sn PCR c p dng chn on bnh, trong bao gm: Bnh tm: MBV, WSSV, YHV, TSVv bnh trn c: VNN

Phn II. Trang thit b v ha cht

1. Trang thit b 1.1. Trm tch trit ADN v RNA Trang thit b cn dng cho trm tch chit ADN v RNA bao gm cc dng c sau: T lnh thng T lnh su My ly tm lnh 13000rpm Block nhit kh Vortex My ht chn khng Cc loi Micropipette 0.5 - 1000l Gi ng ng nghim tube Eppendorf 0.2 1.5ml Hp ng u tp Micropipette cc loi B ko, panh My lm lnh hoc hp lnh Chai c vi ng cn 95% ng nghim tube Eppendorf 0.2 v 1.5ml Tp Micropipette cc loi Gang tay, giy lau, giy thm Bt nh du S ghi chp Chy nghin mu (loi dng mt ln hoc loi c th dng li) 1.2. Trm PCR Cc trang thit b cn thit cho trm PCR bao gm: My PCR (Eppendorf) n p LIOA 10000W
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1.3. Trm phn tch kt qu Trang thit b cn thit cho trm phn tch kt qu bao gm: B in di Bn c UV B chp nh gel My vi tnh L vi sng Cn in t ng ong 100ml, 500ml v 1000ml Bnh tam gic 500ml v 1000ml Bnh tam gic chu nhit 250ml ng nghim 50ml bng nha c np Micropipette 1 - 20l Tp Micropipette Gang tay, giy lau Giy Parafin 2. Ha cht Cc loi ha cht cn dng trong phng chn on tc nhn vi rt gy bnh trn tm, c bao gm cc loi sau: Nc ct Cn 75%, 95% Chloroform Isopropanol Ethidiumbrome Agarose TAE buffer 1X hoc TBE buffer 1X

3. Cc loi Kt thng dng IQ2000VNN Non Stop Nested WSSV PCR kits (Nam Khoa) Duplex MBV WSSV PCR mix (Nam Khoa) IQ2000WSSV IQ2000YHV IQ2000TSV

Phn III. Phng php chn on bnh ng vt Thy sn bng phng php PCR
1. Quy trnh chn on Quy trnh chn on tc nhn vi rt gy bnh trn tm cc c thc hin theo cc bc sau: Quy trnh chn on tc nhn vi rt gy bnh trn tm Mu tm
Chun b mu

Tch trit ADN (MBV v WSV)

Tch trit RNA ( YHV, TSV)

PCR

RT- PCR

in di

c kt qu

Quy trnh chn on VNN trn c bin

Mu c
Chun b mu

Tch trit RNA

RT- PCR

in di

c kt qu

2. Phng php chn on bnh MBV trn tm s Phng php chn on bnh MBV trn tm s bng phng php PCR s dng kt Duplex MBV WSSV ca Cng ty Nam Khoa ch yu c s dng i vi tm Postlarvae nh (< PL8). 2.1. Thnh phn b kt Thng thng b kt Duplex MBV WSSV c 50 mix Dung dch tch chit ADN cho 50 mu: R1 (50ml), R2 (300l) v R3 (150l). Pha dung dch tch chit bng cch dng u tp sch ht 250l R2 vo R1, lc u sau dng tp sch ht 125l R3 cho tip vo R1, lc u ( ch dung dch tch chit pha phi trong ti, thi hn s dng 3 thng k t ngay pha)
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 Duplex MBV WSSV PCR mix: 1 x 50 tube PCR loi 0.2ml, mi ng cha 48l mix (bo qun 200C) i chng dng v i chng m (bo qun 200C) TBE 10X: 2 x50ml Loading buffer: 500l Ethidium bromide10mg/ml: 15l Agarose:3gr Ngoi ra b kt cng cung cp cho 50 b ci chy nghin mu v tube Eppendorf 1.5ml 2.2. Chun b mu Mu l tm Postlarvae, tm ging c nh tin hnh ly khong 50 con Mu tm nui: tin hnh ct mt phn mang, chn bi hoc ui ca 10 15 con (tng lng mu khng qu 1 gr) i vi tm b m tin hnh ct chn bi hoc ui Tt c cc loi mu trn c th ly mu ti hoc c nh trong cn 95% 2.3. Tch chit ADN Cho mu vo Eppendorf 1.5ml, sau thm vo 100l dung dch tch chit, dng chy nghin nh, sau khi nghin nh v nhuyn tin hnh thm vo t 500 700ml dung dch tch chit, sau tip tc nghin nhuyn hon ton ri y np li (nu thc hin nhiu mu mt ln th cn phi lnh trc khi chuyn sang bc tip theo) mu: sau khi nghin xong th tin hnh chuyn vo block nhit kh (iu chnh nhit ca block l 960C) v gi trong khong 5 10 pht, sau ly ra v lm lnh nhanh (trong hoc t lnh su) trong vi pht Ly tm 13000 vng/pht trong 5 pht. S dng phn dch ni chy PCR hoc bo qun trong t lnh su

2.4. Quy trnh tin hnh PCR Chun b phn ng: Cho 2l dung dch tch chit vo 1 ng PCR mix 48l, 2l i chng m 1 ng PCR mix 48l v 2l i chng dng 1 ng PCR mix 48l Thc hin phn ng trong my lun nhit theo 1 trong 2 chng trnh sau ty thuc vo mc ch: Pht hin MBV v sng lc WSSV 01 chu k 400C trong 10 pht 01 chu k 950C trong 5 pht 40 chu k 940C trong 30 giy 490C trong 30 giy 720C trong 1 pht 01 chu k 720C trong 7 pht Gi 40C cho n khi phn tch Hoc Pht hin WSSV v sng lc MBV 01 chu k 400C trong 10 pht 01 chu k 950C trong 5 pht 20 chu k 940C trong 30 giy 580C trong 30 giy 720C trong 1 pht 35 chu k 940C trong 30 giy 510C trong 30 giy 720C trong 1 pht 01 chu k 720C trong 7 pht Gi 40C cho n khi phn tch

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2.5. c kt qu Chun b bn gel: Pha1,5 2%Agarose bng dung dch m TBE 1X hoc TAE 1X vo bnh tam gic 250ml (ly khong 45 50ml dung dch m TAE 1X, cn khong 0,7 1gr Agarose cho vo), sau un cho Agarose tan ht (khng cn bt kh), i n khi nhit gim xung cn khong 600C cho vo khong 2 - 3l Ethidium bromide lc nh Ethidium bromide tan u ri tin hnh vo bn gel ( ch khng nn bn gel qu dy, <0,8cm), Khi bn gel ng li th nh nhng g b cc lc trn bn gel, chuyn bn gel vo mng in di, dung dch m ( cng loi vi dung dch dng un Agarose) vo mng in di cho ti khi ngp bn gel l c. in di: cho vo sn phm in di khong 10 -12l Loading buffer trn u ( hoc dng giy Parafin ri nh khong 2 - 3l Loading buffer, ht sn phn PCR ra nh vo v trn u), sau ly khong 10l nh vo 01 ging trn bn gel. in di hiu in th l 100 volt, trong thi gian 35 40 pht c kt qu: So snh kt qu theo nh sau: C(+) C(-) S1 S2 S3 C(+): i chng dng C(-): i chng m S1: Mu b nhim MBV S2: Mu b nhim WSSV S3: Mu m tnh

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3. Chn on tc nhn gy bnh m trng (WSSV) trn tm s v tm Chn trng Chng ta c th s dng mt trong cc phng php sau: 3.1. Chn on tc nhn gy bnh m trng bng phng php PCR s dng kt Non-Stop Nested WSSV PCR ca cng ty Nam Khoa 2.1. Thnh phn b kt Thng thng b kt Non Stop Nested WSSV c 50 mix Dung dch tch chit ADN cho 50 mu: R1 (50ml), R2 (300l) v R3 (150l). Pha dung dch tch chit bng cch dng u tp sch ht 250l R2 vo R1, lc u sau dng tp sch ht 125l R3 cho tip vo R1, lc u ( ch dung dch tch chit pha phi trong ti, thi hn s dng 3 thng k t ngay pha) Non Stop Nested WSSV PCR mix: 1 x 50 tube PCR loi 0.2ml, mi ng cha 48l mix (bo qun 200C) i chng dng v i chng m (bo qun 200C) TBE 10X: 2 x50ml Loading buffer: 500l Ethidium bromide10mg/ml: 15l Agarose:3gr Ngoi ra b kt cng cung cp cho 50 b ci chy nghin mu v tube Eppendorf 1.5ml 2.2. Chun b mu Mu l tm Postlarvae, tm ging c nh tin hnh ly khong 50 con Mu tm nui: tin hnh ct mt phn mang, chn bi hoc ui ca 10 15 con (tng lng mu khng qu 1 gr) i vi tm b m tin hnh ct chn bi hoc ui Tt c cc loi mu trn c th ly mu ti hoc c nh trong cn 95% 2.3. Tch chit ADN
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 Cho mu vo Eppendorf 1.5ml, sau thm vo 100l dung dch tch chit, dng chy nghin nh, sau khi nghin nh v nhuyn tin hnh thm vo t 500 700ml dung dch tch chit, sau tip tc nghin nhuyn hon ton ri y np li (nu thc hin nhiu mu mt ln th cn phi lnh trc khi chuyn sang bc tip theo) mu: sau khi nghin xong th tin hnh chuyn vo block nhit kh (iu chnh nhit ca block l 960C) v gi trong khong 5 10 pht, sau ly ra v lm lnh nhanh (trong hoc t lnh su) trong vi pht Ly tm 13000 vng/pht trong 5 pht. S dng phn dch ni chy PCR hoc bo qun trong t lnh su 2.4. Quy trnh tin hnh PCR Chun b phn ng: Cho 2l dung dch tch chit vo 1 ng PCR mix 48l, 2l i chng m 1 ng PCR mix 48l v 2l i chng dng 1 ng PCR mix 48l Thc hin phn ng trong my lun nhit theo chng trnh sau: 01 chu k 400C trong 10 pht 01 chu k 950C trong 5 pht 20 chu k 940C trong 30 giy 580C trong 30 giy 720C trong 1 pht 35 chu k 940C trong 30 giy 510C trong 30 giy 720C trong 1 pht 01 chu k 720C trong 7 pht Gi 40C cho n khi phn tch

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2.5. c kt qu Chun b bn gel: Pha1,5 2%Agarose bng dung dch m TBE 1X hoc TAE 1X vo bnh tam gic 250ml (ly khong 45 50ml dung dch m TAE 1X, cn khong 0,7 1gr Agarose cho vo), sau un cho Agarose tan ht (khng cn bt kh), i n khi nhit gim xung cn khong 600C cho vo khong 2 - 3l Ethidium bromide lc nh Ethidium bromide tan u ri tin hnh vo bn gel ( ch khng nn bn gel qu dy, <0,8cm), Khi bn gel ng li th nh nhng g b cc lc trn bn gel, chuyn bn gel vo mng in di, dung dch m ( cng loi vi dung dch dng un Agarose) vo mng in di cho ti khi ngp bn gel l c. in di: cho vo sn phm in di khong 10 -12l Loading buffer trn u ( hoc dng giy Parafin ri nh khong 2 - 3l Loading buffer, ht sn phn PCR ra nh vo v trn u), sau ly khong 10l nh vo 01 ging trn bn gel. in di hiu in th l 100 volt, trong thi gian 35 40 pht c kt qu: So snh kt qu theo nh sau: C(+) C(-) S1 S2 S3 S4 C(+): i chng dng C(-): i chng m S1: Mu b nhim WSSV mc nng S2 v S3: Mu b nhim WSSV mc nh S4: Mu khng b nhim WSSV

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3.2. Chn on tc nhn gy bnh bng phng php PCR s dng kt IQ2000WSSV 3.2.1. Thnh phn B kt c cung cp cho 200 mu, bao gm cc thnh phn nh sau: First PCR PreMix 4 ng, mi ng cha 450l (Bao gm dung dch phn ng, dNTP v primer chuyn bit) Nested PCR PreMix 4 ng, mi ng cha 840l (Bao gm dung dch phn ng, dNTP v Primer chuyn bit) i chng dng (Positive standard) 1 ng, cha 100l, 104 bn sao/l i chng m Thang ADN 333bp) Dung dch tch chit ADN ( Lysis buffer) 1 chai 100ml 3.2.3. Chun b mu ( xem phn chun b mu kt Duplex MBV WSSV) 3.2.3. Tch chit AND Cho mu vo ng Eppendorf 1.5ml ( nu mu c bo qun trong cn th cn lm kh cn mu trc khi a vo tch chit) Cho vo ng Eppendorf c cha mu khong 100 - 200l dung dch tch chit, nghin nh v mn bng chy nghin, sau thm vo khong 400 500l dung dch tch chit v nghin tip n khi nhuyn. mu nhit 950C trong 10 pht, ly tm 12000 vng/pht trong thi gian 10 pht Chuyn 200l phn dch ni sang 1 ng Eppendorf mi c cha 400l cn 95%, lc nh nhng Ly tm 12000 vng/pht trong thi gian 5 pht, sau loi b cn v lm kh mu Ha tan ADN bng nc tinh khit vi lng khong t 50 - 200l ty thuc vo lng ADN tch chit c
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1 ng, cha 100l ( thang ADN c 3 vch, 848bp, 630bp v

3.2.4. Khuch i sn phm iu kin phn ng - Bc 1 940C 30 giy ; 620C 30 giy; 720C 30 giy, lp li 5 chu k, sau 940C 15 giy; 620C 15 giy; 720C 20 giy, lp li 15 chu k, sau 720C 30 giy; 200C 30 giy, kt thc chu k cui - Bc 2 940C 20 giy; 620C 20 giy; 720C 30 giy, lp li 25 chu k, Add 720C 30 giy; 200C 30 giy, kt thc chu k cui Chun b phn ng + Chun b cc cht phn ng nh sau: Bc 1 : 8l/phn ng, bao gm cc cht - First PCR PreMix 7,5l - IQzyme ADN Polymerase 2U/l 0,5l Bc 2: 15l/ phn ng, bao gm cc cht - Nested PCR Premix 14l

- IQzyme ADN Polymerase 2U/l 1l + Quy trnh thc hin nh sau: - Chun b hn hp phn ng bc 1 v 2 theo s mu chn on, 1 mu i chng dng v 1 mu i chng m, trn chung vo 1 ng Eppendorf - Cho 8l hn hp phn vo ng Eppendorf 0,2ml, ghi k hiu mu ln ng Eppendorf - Cho vo mi ng Eppendorf c cha hn hp phn ng 2l dung dch ADN mu tch chit, i chng m v dng - y np li
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- Tin hnh khuch i sn phm bc 1 trong my PCR - Sau khi kt thc qu trnh khuch i sn phm bc 1, thm vo 15l hn hp phn ng bc 2 - Tin hnh khuch i sn phm bc 2 3.2.5. in di Quy trnh tin hnh theo nguyn l chung ca in di. Tin hnh hiu in th 100volt trong thi gian 25 30 pht 3.2.6. c kt qu c kt qu da trn hnh chun sau

Trong 1: Mu b nhim WSS mc nng 2: Mu b nhim WSS mc trung bnh 3: Mu b nhim WSS mc nh 4: Mu b nhim WSS mc rt nh 5: Mu khng b nhim WSSV 6: Mu nc tinh khit 7: i chng dng 2000 bn sao 8: i chng dng 200 bn sao 9: i chng dng 20 bn sao M: Thang ADN 848bp, 630bp v 333bp Quy trnh chn on
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- Nu mu kim tra xut hin vch 296bp v 550bp hoc ch c vch 550bp th mu b nhim WSSV - Nu mu kim tra ch xut hin vch 848bp th mu khng b nhim WSSV Ngoi ra ta cng c th s dng kt Duplex MBV WSSV cho vic chn on tc nhn gy bnh m trng trn tm s v tm Chn trng ( Xem phn Quy trnh chn on bnh MBV) 4. Chn on bnh u vng trn tm s bng phng php RT PCR s dng kt IQ2000YHV/GAV 4.1. Thnh phn B kt c cung cp cho 200 mu, bao gm cc thnh phn nh sau: RT - PCR PreMix 4 ng, mi ng cha 420l (Bao gm dung dch phn ng, dNTP v Primer YHV/GAV chuyn bit) Nested PCR PreMix 4 ng, mi ng cha 840l (Bao gm dung dch phn ng, dNTP v Primer YHV/GAVchuyn bit) i chng dng (YHV v GAV ) 2 ng, cha 100l, 104 bn sao/l i chng m (Yeast tRAN) 1 ng 500l, 40ng/l IQzyme ADN Polymerase 1 ng 2U/l, cha 360l RT Enzyme Mix Thang ADN 333bp) Dung dch tch chit RNA 1 chai 100ml Nc tinh kit 1 chai 100ml 4.2. Chun b mu ( xem phn chun b mu kt Duplex MBV WSSV) 4.3. Tch chit RNA Cho mu vo ng Eppendorf 1.5ml ( nu mu c bo qun trong cn th cn lm kh cn mu trc khi a vo tch chit)
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1 ng, cha 12l

Dung dich nhum (6X Loading buffer Dye) 1 ng, cha 1500l 1 ng, cha 100l ( thang ADN c 3 vch, 848bp, 630bp v

 Cho vo ng Eppendorf c cha mu khong 100 - 200l dung dch tch chit, nghin nh v mn bng chy nghin, sau thm vo khong 400 500l dung dch tch chit v nghin tip n khi nhuyn. nhit phng trong thi gian khong 5 pht Cho vo 100l CHCl3 (Chloroform), lc trn my Vortex khong 20 giy, nhit phng trong khong 3 pht ri ly tm 12000 vng/pht trong thi gian 15 pht Chuyn 200l phn dch ni sang 1 ng Eppendorf mi c cha 200l Isopropanol, lc nh nhng Ly tm 12000 vng/pht trong thi gian 10 pht, sau loi b Isopropanol Ra vi 500l cn 75%, ly tm 9000 vng/pht trong thi gian 5 pht Loi b cn v lm kh Ha tan RNA bng nc tinh khit vi lng khong t 50 - 200l ty thuc vo lng RNA tch chit c 4.4. Khuch i sn phm iu kin phn ng - Bc 1 420C 30 pht ; 940C 2 pht, sau 940C 20giy; 620C 20 giy; 720C 30 giy, lp li 15 chu k, sau 720C 30 giy; 200C 30 giy, kt thc chu k cui - Bc 2 940C 20 giy; 620C 20 giy; 720C 30 giy, lp li 30 chu k 720C 30 giy; 200C 30 giy, kt thc chu k cui Chun b phn ng + Chun b cc cht phn ng nh sau:
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Bc 1 : 8l/ hn hp phn ng, bao gm cc cht - RT - PCR PreMix 7l - IQzyme ADN Polymerase 2U/l 0,5l - RT Enzyme Mix 0,5l

Bc 2: 15l/ phn ng, bao gm cc cht - Nested PCR Premix 14l

- IQzyme ADN Polymerase 2U/l 1l + Quy trnh thc hin nh sau: - Chun b hn hp phn ng bc 1 v 2 theo s mu chn on, 1 mu i chng dng v 1 mu i chng m, trn chung vo 1 ng Eppendorf - Cho 8l hn hp phn vo ng Eppendorf 0,2ml, ghi k hiu mu ln ng Eppendorf - Cho vo mi ng Eppendorf c cha hn hp phn ng 2l dung dch RNA mu tch chit, i chng m v dng - y np li - Tin hnh khuch i sn phm bc 1 trong my PCR - Sau khi kt thc qu trnh khuch i sn phm bc 1, thm vo 15l hn hp phn ng bc 2 - Tin hnh khuch i sn phm bc 2 4.5. in di Quy trnh tin hnh theo nguyn l chung ca in di. Tin hnh hiu in th 100volt trong thi gian 25 30 pht 4.6. c kt qu c kt qu da trn hnh chun sau

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Trong 1: i chng dng YHV, 2000 bn sao 2: i chng dng YHV, 200 bn sao 3: i chng dng YHV, 20 bn sao 4: i chng dng GAV, 2000 bn sao 5: i chng dng GAV, 200 bn sao 6: i chng dng GAV, 20 bn sao 7: Mu b nhim YHV mc nng 8: Mu b nhim YHV mc nh 9: Mu b nhim GAV mc nng 10: Mu b nhim GAV mc nh 11: i chng m M: Thang ADN, 848bp, 630bp v 333bp Quy trnh chn on - Nu mu kim tra xut hin vch 277bp v 777bp th mu b nhim YHV mc nng - Nu mu kim tra ch xut hin vch 277bp th mu b nhim YHV mc nh - Nu mu kim tra xut hin vch 406bp v 777bp th mu b nhim GAV mc nng

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- Nu mu kim tra ch xut hin vch 406bp th mu b nhim GAV mc nh - Nu ch xut hin vch 680bp th mu khng b nhim YHV v GAV 5. Chn on hi chng Taura bng phng php RT- PCR s dng kt IQ2000TSV 5.1. Thnh phn B kt c cung cp cho 200 mu, bao gm cc thnh phn nh sau: RT - PCR PreMix 4 ng, mi ng cha 420l (Bao gm dung dch phn ng, dNTP v Primer TSV chuyn bit) Nested PCR PreMix 4 ng, mi ng cha 840l (Bao gm dung dch phn ng, dNTP v Primer TSVchuyn bit) i chng dng (TSV ) 1 ng, cha 100l, 104 bn sao/l i chng m (Yeast tRAN) 1 ng 500l, 40ng/l IQzyme ADN Polymerase 1 ng 2U/l, cha 360l RT Enzyme Mix Thang ADN 333bp) Dung dch tch chit RNA 1 chai 100ml Nc tinh kit 1 chai 100ml 5.2. Chun b mu ( xem phn chun b mu kt Duplex MBV WSSV) 5.3. Tch chit RNA Cho mu vo ng Eppendorf 1.5ml ( nu mu c bo qun trong cn th cn lm kh cn mu trc khi a vo tch chit) Cho vo ng Eppendorf c cha mu khong 100 - 200l dung dch tch chit, nghin nh v mn bng chy nghin, sau thm vo khong 400 1 ng, cha 12l Dung dich nhum (6X Loading buffer Dye) 1 ng, cha 1500l 1 ng, cha 100l ( thang ADN c 3 vch, 848bp, 630bp v

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500l dung dch tch chit v nghin tip n khi nhuyn. nhit phng trong thi gian khong 5 pht Cho vo 100l CHCl3 (Chloroform), lc trn my Vortex khong 20 giy, nhit phng trong khong 3 pht ri ly tm 12000 vng/pht trong thi gian 15 pht Chuyn 200l phn dch ni sang 1 ng Eppendorf mi c cha 200l Isopropanol, lc nh nhng Ly tm 12000 vng/pht trong thi gian 10 pht, sau loi b Isopropanol Ra vi 500l cn 75%, ly tm 9000 vng/pht trong thi gian 5 pht Loi b cn v lm kh Ha tan RNA bng nc tinh khit vi lng khong t 50 - 200l ty thuc vo lng RNA tch chit c 5.4. Khuch i sn phm iu kin phn ng - Bc 1: RT - PCR 420C 30 pht ; 940C 2 pht, sau 940C 20giy; 620C 20 giy; 720C 30 giy, lp li 15 chu k, sau 720C 30 giy; 200C 30 giy, kt thc chu k cui - Bc 2: Nested PCR 940C 20 giy; 620C 20 giy; 720C 30 giy, lp li 30 chu k 720C 30 giy; 200C 30 giy, kt thc chu k cui Chun b phn ng + Chun b cc cht phn ng nh sau: Bc 1 : 8l/ hn hp phn ng, bao gm cc cht - RT - PCR PreMix 7l
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- IQzyme ADN Polymerase 2U/l 0,5l - RT Enzyme Mix 0,5l

Bc 2: 15l/ phn ng, bao gm cc cht - Nested PCR Premix 14l

- IQzyme ADN Polymerase 2U/l 1l + Quy trnh thc hin nh sau: - Chun b hn hp phn ng bc 1 v 2 theo s mu chn on, 1 mu i chng dng v 1 mu i chng m, trn chung vo 1 ng Eppendorf - Cho 8l hn hp phn vo ng Eppendorf 0,2ml, ghi k hiu mu ln ng Eppendorf - Cho vo mi ng Eppendorf c cha hn hp phn ng 2l dung dch RNA mu tch chit, i chng m v dng - y np li - Tin hnh khuch i sn phm bc 1 trong my PCR - Sau khi kt thc qu trnh khuch i sn phm bc 1, thm vo 15l hn hp phn ng bc 2 - Tin hnh khuch i sn phm bc 2 5.5. in di Quy trnh tin hnh theo nguyn l chung ca in di. Tin hnh hiu in th 100volt trong thi gian 25 30 pht 5.6. c kt qu c kt qu da trn hnh chun sau

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Trong 1: i chng dng, 2000 bn sao 2: i chng dng, 200 bn sao 3: i chng dng, 20 bn sao 4: i chng m 5: Mu b nhim TSV nng 6: Mu b nhim TSV mc trung bnh 7: Mu b nhim TSV mc nh 8: Mu b nhim TSV mc rt nh 9: Mu khng b nhim TSV M: Thang ADN, 848 bps, 630 bps, 333 bps Quy trnh chn on - Nu mu kim tra xut hin vch 284bp v 476bp th mu b nhim TSV mc nng - Nu mu kim tra ch xut hin vch 284bp th mu b nhim TSV mc nh - Nu ch xut hin vch 680bp th mu khng b nhim TSV

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5. Chn on bnh VNN trn c bin bng phng php RT- PCR s dng kt IQ2000VNN 5.1. Thnh phn B kt c cung cp cho 200 mu, bao gm cc thnh phn nh sau: RT - PCR PreMix 4 ng, mi ng cha 420l (Bao gm dung dch phn ng, dNTP v Primer VNN chuyn bit) Nested PCR PreMix 4 ng, mi ng cha 840l (Bao gm dung dch phn ng, dNTP v Primer VNN chuyn bit) i chng dng (VNN ) 1 ng, cha 100l, 104 bn sao/l i chng m (Yeast tRAN) 1 ng 500l, 40ng/l IQzyme ADN Polymerase 1 ng 2U/l, cha 360l RT Enzyme Mix Thang ADN 333bp) Dung dch tch chit RNA 1 chai 100ml Nc tinh kit 1 chai 100ml 5.2. Chun b mu ( xem phn chun b mu kt Duplex MBV WSSV) 5.3. Tch chit RNA Cho mu vo ng Eppendorf 1.5ml ( nu mu c bo qun trong cn th cn lm kh cn mu trc khi a vo tch chit) Cho vo ng Eppendorf c cha mu khong 100 - 200l dung dch tch chit , nghin nh v mn bng chy nghin, sau thm vo khong 400 500l dung dch tch chit v nghin tip n khi nhuyn. nhit phng trong thi gian khong 5 pht 1 ng, cha 12l Dung dich nhum (6X Loading buffer Dye) 1 ng, cha 1500l 1 ng, cha 100l ( thang ADN c 3 vch, 848bp, 630bp v

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 Cho vo 100l CHCl3 (Chloroform), lc trn my Vortex khong 20 giy, nhit phng trong khong 3 pht ri ly tm 12000 vng/pht trong thi gian 15 pht Chuyn 200l phn dch ni sang 1 ng Eppendorf mi c cha 200l Isopropanol, lc nh nhng Ly tm 12000 vng/pht trong thi gian 10 pht, sau loi b Isopropanol Ra vi 500l cn 75%, ly tm 9000 vng/pht trong thi gian 5 pht Loi b cn v lm kh Ha tan RNA bng nc tinh khit vi lng khong t 50 - 200l ty thuc vo lng RNA tch chit c 5.4. Khuch i sn phm iu kin phn ng - Bc 1 420C 30 pht ; 940C 2 pht, sau 940C 30giy; 620C 30 giy; 720C 30 giy, lp li 15 chu k, sau 720C 30 giy; 200C 30 giy, kt thc chu k cui - Bc 2 940C 20 giy; 620C 20 giy; 720C 30 giy, lp li 30 chu k 720C 30 giy; 200C 30 giy, kt thc chu k cui Chun b phn ng + Chun b cc cht phn ng nh sau: Bc 1 : 8l/ hn hp phn ng, bao gm cc cht - RT - PCR PreMix 7l - IQzyme ADN Polymerase 2U/l 0,5l

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- RT Enzyme Mix

0,5l

Bc 2: 15l/ phn ng, bao gm cc cht - Nested PCR Premix 14l

- IQzyme ADN Polymerase 2U/l 1l + Quy trnh thc hin nh sau: - Chun b hn hp phn ng bc 1 v 2 theo s mu chn on, 1 mu i chng dng v 1 mu i chng m, trn chung vo 1 ng Eppendorf - Cho 8l hn hp phn vo ng Eppendorf 0,2ml, ghi k hiu mu ln ng Eppendorf - Cho vo mi ng Eppendorf c cha hn hp phn ng 2l dung dch RNA mu tch chit, i chng m v dng - y np li - Tin hnh khuch i sn phm bc 1 trong my PCR - Sau khi kt thc qu trnh khuch i sn phm bc 1, thm vo 15l hn hp phn ng bc 2 - Tin hnh khuch i sn phm bc 2 5.5. in di Quy trnh tin hnh theo nguyn l chung ca in di. Tin hnh hiu in th 100volt trong thi gian 25 30 pht 5.6. c kt qu c kt qu da trn hnh chun sau

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Trong 1: i chng chun 1, 2000 bn sao/ phn ng 2: i chng chun 2, 200 bn sao/phn ng 3: i chng chun 3, 20 bn so/phn ng 4: Mu nc tinh khit 5: Mu b nhim VNN mc nng 6: Mu b nhim VNN mc nh 7: Mu khng b nhim VNN M: Thang ADN, 848 bps, 630 bps, 333 bps Quy trnh chn on - Nu mu kim tra ch xut hin 1 vch 289bp th mu b nhim VNN mc nh - Nu mu kim tra xut hin vch 289 v 620bp th mu b nhim VNN mc trung bnh - Nu mu kim tra xut hin vch 289, 620 v 1160bp th mu b nhim VNN mc nng

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