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Bo co thc hnh SHPT Nhm 1.4 Lp 50 CNSH

BI BO CO THC HNH SINH HC PHN T:CHN ON BNH VIRUS TRN NG VT THY SN

( VIRUS WSSV GY BNH M TRNG TM)

I. Gii thiu v bnh m trng

Bnh m trng l loi dch bnh nguy him, gy ri ro nht cho nginui tm s t trc n nay (c pht hin t nm 1993). Bnh do mt loivirus c tn l Virus gy hi chng m trng (tn ting Anh l white spotsyndrome virus). Khi xm nhp vo tm, virus s c tr nhiu b phn catm nh m d dy, mang, trng, mt, chn bi, Cc virus ny s sinh snrt nhanh lm tm nhim bnh nng v sau tip tc pht tn ra mi trng

bn ngoi gy bnh cho c n tm c trong ao. T nhng thng tin trn, chothy mc nguy him ca bnh m trng l rt cao.

Virus gy hi chng m trng (WSSV) l mt trong nhng nhm virusc xp vo nhm virus gy cht cp tnh trn tm nui. WSSV gy cht trnhu ht cc loi tm thuc nhm Penaeus nh P. monodon, P. japonicus,P.chinensis, P. merguiensis v trn tt c cc giai on pht trin ca tm tu trng n tm ging, tm trng thnh. Hnh thi

V WSSV l nhng tiu phn i xng, c hnh ellipse hoc hnh que,khng to th n, ng knh t 120 150 nm, chiu di t 270 290 nm.WSSV c mt phn ph ging nh ui im cui ca v. Mt s loinucleocapsid c bit c ng knh t 65 70 nm, chiu di t 300 350 nm.

Virus WSSV mang vt cht di truyn l DNA.

Hnh 1: Hnh thi ca virus m trng quan st di knh hin vi in t(A): phn v (B ): nuclocapsid

II. Phng php.1. Gii thiu s lc v PCR

Trong sinh hc phn t c nhiu phng php khch i invitro c chn lcnhng phng php ni ting nht, c ph bin nhiu nht l PCR

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(Polymerase Chain Reaction).PCR do Karl Mullis v cng s pht minh vonm 1985 v to bc nhy vt trong sinh hc phn t v k thut gene.

Hnh 2: my PCR Nguyn tc ca PCR l da trn hot ng ca DNA polymerase trong qu

trnh tng hp DNA mi t mch khun v kh nng bin tnh v hi tnh caDNA

PCR l phn ng nhn bn on DNA nh enzyme polymerease in vitro v primer, trong vi gi c th to ra 230-235 bn sao t mt trnh t DNA, lmt k thut ph bin trong sinh hc phn t nhm khuych i mt onDNA m khng cn s dng cc sinh vt sng.

PCR c s dng lm tng tc khm ph b gene ngi v khm ph sm cc bnh virus. PCR lm thun li cho vic to dng cc trnh t DNAv to thnh mt c s t nhin cho vic gii trnh t bng phng php Sanger v nhiu k thut sinh hc phn t khcCng vi PCR , RT-PCR cng tr thnh mt k thut c p dng rng ri nht trong nghin cu sinh y, RT-PCR to ra nhiu c hi trong chun on, cho php pht hin cc viusRNA.2. Chng trnh PCR

PCR l mt chui nhiu chu k (cycle) ni tip nhau. Mi chu k gm 3giai on:

GIAI ON 1: BIN TNHNhit ca ng mu s c tng ln n 94-95oC trong vng 30s n 1

pht, nhit ny s lm cho phn t DNA bin tnh tch ra lm i honton, tt c hot ng ca cc enzyme u dng li (V d hot ng ko di chu k trc). Mc ch ca giai on ny lm cho phn t DNA tch ra lmDNA mch i v c dui thng ra hon ton, t d dng cho vic bt cp

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http://vi.wikipedia.org/wiki/T%E1%BA%ADp_tin:Pcr_machine.jpg


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v gn cc Nu vo mch n theo nguyn tc b sung.

GIAI ON 2: BT CPNhit c h thp (thp hn Tm ca cc mi) cho php cc mi bt

cp vi khun, thng dao ng t 40- 70 oC .Cc Primer (on mi) c sntrong ng mu s bt cp vo cc chui n DNA sau khi bin tnh theonguyn tc b sung.

S bt cp ny s c 2 ngha c hiu: c hiu theo nguyn tc b sung vi on gen m ta cn khuch i c hiu theo chiu di ca on gen khuch i.

GIAI ON 3 : KO DINhit c tng n 72oC gip cho DNA polymerase s dng vn l

polymerase chu nhit hot ng tt nht. Enzyme ny s gn cc Nu c sntrong ng nghim vo cc primer theo nguyn tc b sung, t lm cho onmi di ra v hnh thnh nn mt mch n mi b sung vi mch n c. Vmt bn sao DNA mi c hnh thnh. Nh vy, sau giai on ny ta cc hai phn t DNA c cu trc ging ht nh phn t DNA ban u.

C ba giai on bin tnh, bt cp v ko di thuc mt chu k ca khuch

i DNA. Nh vy, sau mi chu k khuch i th t mt phn t DNA banu s thnh 2 phn t DNA ging ht nhau, v sau 30 chu k khuch i s ckhong 230 phn t DNA ging ht nhau (Khong 1 t DNA). Nh vy khi slng DNA trong mu l vi triu th ch sau 30 chu k khuch i (Thi giankhong 2-3 gi) ta c hng nghn t bn sao, lng DNA ny ta lm phn ng ct cho gii trnh t chui.

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3. Nhng vn cn ch trong k thut PCR. 3.1. Hot start ( khi u nhit cao)

Khi ng nng PCR (hot start) l phng php sn xut cc sn phmPCR sch hn. DNA khun mu v primer c trn vi nhau v c gi nhit trn ngng ca lin kt khng c hiu gia primer v khun mu.Tt c cc thnh phn ca PCR c b sung trc ngoi tr mt cht thencht thng l (Taq pol). Ch trc khi i vo chu k khch i, thnh phncha c mi c b sung cho php phn ng xy ra nhit cao hn. Dokhng c hin tng lai khng c hiu gia primer vi khun mu, nn cc bng DNA c khch i tr nn sng hn, cc on primer khng c c hi gn li vi nhau.

3.2. EnzymeVic chn DNA polymerease c xc nh bi mc ch ca th

nghim. C hng lot enzyme thng mi sn c la chn, Taq DNA polymerase l c s dng ph bin nht v c nghin cu rng ri nht.Lng enzyme cn thit l ph thuc vo lng DNA template v kch thc phn ng PCR.

3.3. BuffterHiu sut, tnh chnh xc ca enzyme DNA polymerase ph thuc nhiu

vo h buffer s dng. Thng thng nn s dng ng loi buffer tng ngvi enzyme do nh sn xut khuyn co.

Nhiu loi buffer c sn mt lng nht nh ion Mg2+ nn cn lu khiti u ha nng Mg2+

3.4. Nng Mg2+ v dNTP- Vic tng nng ion Mg2+ c th lm xut hin thm cc sn phm

khng c hiu. Tuy nhin nu thiu ion Mg2+ th dn n lng sn phm PCR thp.

- Nng ion Mg2+ ti u ph thuc vo loi DNA polymerase, loiDNA template v thnh phn buffer (mt s buffer thng mi csn 1 lng nht nh ion Mg2+).

- D tm nng Mg2+ ti u thng thng bng gradien nng t 0.5n 2 mM vi cc khong cch mi bc l 0.2 mM.- dNTPs l loi ha cht km bn cn lu khi s dng v bo qun. Nn

chia nh lng dNTPs gim s ln lm tan, s dng li.- Lng dNTP ti u l 200 M of mi loi dNTP. Vic tng dNTP

khng lm tng ng k lng sn phm PCR so vi vic hiu chnh ccyu t khc.

3.5. Primers

Mi c thit k b sung vi trnh t u cui ca on DNA ch PCR,mi thng di khong 15-25 nucleotide v c khong 50-60% G +C. Mi cn

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c hiu vi DNA khun v trnh cc trnh t kp tc, trnh t kt thc PCR t c hiu qu.III. Vt liu, thit b v ha cht. 1. Mu vt th nghim

- 3 i chn bi tm (mua ngoi th trng)2. Dng c, thit b

- My PCR lun nhit- My li tm lnh- B in di- B chp hnh gel- L vi sng- Cn in t- ng li tm 1,5ml- u tip cc loi- Bt nh du- Ko, kp, panh- Bnh tam gic chu nhit- Cc thy tinh

3. Ha cht:- Cn 95%,75%- Agarose- Nc ct- Ethidium bromide.- TBE buffer.- Choloroform.- Isopropanol.

IV.Phng php thc hin chun on hi chng m trng tm bngKit IQ2000WSSV

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Hnh 3: Qui trnh chn on bnh virus m trng tm s bng k thut PCR

1. Thnh phn ca Kit- Buffer ly gii (200 phn ng).- Kit khuch i trnh t c hiu WSSV (200 phn ng):

+ First PCR Premix.+ Nested PCR Premix.+ P(+) standard.+ tRNA nm men+ IQzyme DNA polymerase+ 6X loading dye

- Thang chun DNA (848bp, 630bp v 333bp)2. Quy trnh tch chit DNA t tm.

- Dng ko ct 3 i chn bi ca tm.- Thm 500l dd m ly gii lysis buffer vo ng 1,5ml- t mu tm vo ng v nghin

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- mu chun b 95o C trong 10 pht, sau em ly tm 12000g(12000 vng/pht, r=5-7cm) trong 10 pht.

- Chuyn 200l dd trong lp trn vo ng 1,5ml sch c 400l cn 95 % , trong 10-15 pht.

- Vortex nhanh, ly tm 12 000 vng trong 5 pht, sau gn b cn vlm kh cn

- Ha tan cn bng 50l nc ct hoc buffer TE. Gii thch:

- m ly gii lysis buffer: ph v mng t bo, mng nhngii phngDNA ra mi trng ng thi phn hy cc protein lin kt vi DNA. Ngoi ra trong m lysis buffer cn cha dd m EDTA gip duy tr pH nh trong mi trng t bo gi cho DNA khng b phn hy.

- Nghin c hc: cng vi m lysis buffer gip ph v cc cu trc t bo(mng t bo, mng nhn,) gii phng DNA.- Ly tm ln 1:da trn s khc bit v kch thc, trng lng phn tch

cc phn t nucleotic v cc cht hu c nh protein, lipid, thnh cc pha khc nhau.

- B sung cn 900 : nhm kt ta DNA- Ly tm ln 2: thu kt ta DNA.- B sung thm m TE:gip gi v bo qun DNA trong mt thi gian di.

m TE c tc dng n nh pH trnh s phn hy cc DNA. Ngoi ram TE cn lin kt vi mt s ion kim loi (Mg2+,..) lm bt hot ccenzyme thy phn nhm gi v bo qun DNA .

3. Quy trnh PCR Chu trnh bc 1 PCR:

5 chu k: 94o C --- 30s62 oC --- 30s

72 oC --- 30s

15 chu k: 94oC --- 15s62 oC --- 15s72 oC --- 20s

Chu k cui: 72oC --- 30s

20o

C --- 30s

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Chng trnh Nested PCR:25 chu k: 92oC --- 20s

62 oC --- 20s

72o

C --- 30s

Chu k cui: 72oC --- 30s20 oC --- 30s

Chun b hn hp phn ng cho bc 1 PCR:8l/phn ng: - Frist PCR Premix 7.5l

- IQzyme DNA Polymerase 2U/l 0.5lChun b hn hp phn ng cho nested PCR:

15l/phn ng: - Nested PCR Premix: 14l- IQzyme DNA polymerase 2U/l 1l

Quy trnh thc hin:- Chun b hn hp phn ng bc 1 v nested PCR cho cc mu v chng

dng (103,102 v 101) chng m (nc hoc tRNA nm men).- Ht 8l hn hp phn ng PCR bc 1 vo ng 0,2ml- Thm 2l DNA khun mu tch chit hoc chng dng vo mi phnng.

- Ht 7,5l frist PCR, trn 0,5l enzyme- Thc hin chng trnh PCR bc 1- Thm 14l hn hp phn ng nested PCR vo mi ng sau khi PCR bc

1 kt thc- Tin hnh Nested PCR - Sau khi phn ng Nested kt thc, thm 5l dd 6X loading dye vo ming phn ng v mix u.

- Sau khi trn u mu c dng cho in di.Gii thch:

Tin hnh PCR thnh 2 bc t c hiu qu cao hn v trong 1 bc s chu k khng c vt qu 40 do: nguyn liu cn kit ,ccon DNA n khng bt cp vi cc on mi m bt cp vi nhau ,xuthin cc yu t ph nh hng n hiu qu PCR

Mt bc gm nhiu chu k, mi chu k u gm 3giai on:bin tnh92oC, bt cp 62oC, ko di 72oC.

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5 chu k u: nguyn liu cn nhiu, s lng DNA cn t, t chu s nhhng ca cc yu t ph nn c th tin hnh trong thi gian lu mi bc l 30s

15 chu k tip theo: hiu qu km hn 5 chu k u nn thi gian mi

bc gim xung ch cn 20s Chu k cui: ko di nhng on DNA cn cha c tng hp xong

v gi 20oC n nh cc DNA va c tng hp. Nested PCR : l mt chin thut gip cho PCR t c kt qu tt hn.

Hn hp phn ng nested PCR gip hn ch s nh hng ca cc yu t ph sau khi tin hnh PCR bc 1, nng enzyme cao hn gip cho phn ng xy ra tt hn.

4.in di.

4.1. Chun b gel agarose.- Chun b dd m TAE hoc TBE. Sau pha long dd in di v 1X s dng cho in di v pha gel agrarose. Ch dd in di v dd to gelagarose phi ging nhau.

- Chun b gel agarose 2%: thm 2g agarose vo bnh c cha 100ml ddin di.

- un hn hp cho ti khi agarose tan ht.- ngui gel agarose trong nhit phng cho ti khi nhit khong 50

oC v t t gel vo khun. dy ca gel khng t hn 0.8cm.4.2. in di.

- t gel agarose vo b in di. DNA s di chuyn v pha cc dng vDNA mang in m.

- Thm dd in di 1X vo b in di cho n khi ph ton b gel.- Load 5-10l hn hp loading dye v sn phm PCR vo mi ging. hn

hp s nm y ging.- Load 5l thang chun ca DNA

- Khi tt c cc mu load xong, lp b in di vi cc in cc v ngunin, bt my 100V- 150V- Kt thc in di, chp hnh gel phn tch kt qu.

5. Kt qu. Khng c kt qu.6. Phn tch nguyn nhn. 6.1. Thao tc k thut

Thao tc cn cha c thnh thc:

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- Qu trnh nghin mu cha tt, lm DNA cha c gii phng honton, DNA b t gy dn n cht lng khng tt.

- Vortex mnh lm DNA b t gy.- Thao tc trong in di cn cha ng k thut

6.2. Thnh phn phn ng.Thnh phn phn ng cha ph hp hoc c th do cc nguyn nhn sau:

- [dNTP] qu cao hoc qu thp- Thnh phn GC trong sn phm- Mu b h hi hay c ln c ch- Primer khng tinh sch- Nng mu qu thp- dNTP khng tinh sch- [primer] qu cao- [enzyme] qu thp- Nc dng cho phn ng khng tinh sch

6.3. Thi gian v nhit bt cp.C th cha ph hp:- Thi gian bin tnh qu di hoc qu ngn- Thi gian bt cp v ko di qu ngn- Qu t chu k phn ng- Nhit bt cp qu cao- Nhit bin tnh qu thp

7. Mt s trng hp khng thnh cng trong PCR.

7.1. Trng hp 1: khng c tn hiu hoc tn hiu vch rt thp.Nguyn nhn:-Thi gian v nhit bt cp:

Qu t chu k phn ngThi gian bt cp v ko di qu ngnNhit bt cp qu caoNhit bin tnh qu thpThi gian bin tnh qu di hoc qu ngn

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- Thnh phn phn ng:[dNTP] qu cao hoc qu thpThnh phn GC trong sn phm cao (>65%)

Mu b h hi hay c ln cht c chPrimer khng tinh sch Nng mu qu thpdNTP khng tinh sch[primer] qu cao[enzyme] qu thpTrnh t mi b sai

Trnh t ch qu di Nc dng cho phn ng khng tinh sch[Mg2+] khng tinh sch

- Thiu thnh phn phn ng:Thiu dNTPThiu primer Thiu mu

Thiu enzymeThiu Mg2+

7.2. Trng hp 2: Sn phm khng c hiu hoc primer- dimerNguyn nhn

- Thi gian bt cp v nhit cha ti u:Qu nhiu chu k phn ngThi gian ko di qu di

Thi gian bt cp qu di hoc qu ngnTc gia gim nhit ca my PCR qu chm Nhit d nng chy ca primer sai

- Thnh phn phn ng PCR cha ti uPrimer khng tinh sch Nng mi qu caoMi c thit k sai

dNTP khng tinh sch

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[Mg2+] qu cao Nc khng tinh sch

7.3. Trng hp 3: to ra cc vt tn hiu

Nguyn nhn:- Thi gian bt cp cha ti u

Qu nhiu chu k phn ngThi gian ko di qu diThi gian bt cp qu di hoc qu ngnTc gia gim nhit ca my PCR qu chm Nhit d nng chy ca primer sai

- Thnh phn phn ng PCR cha ti uPrimer khng tinh schQu nhiu DNA bn muMu b h hiMi c thit k saidNTP khng tinh sch Nc khng tinh sch

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