GVHD:

GVHD:

PCR TRONG CHN ON VIRUS BNH M TRNG TRN TM

CHNG 1. TNG QUAN V BNH M TRNG V CC PHNG PHP PHT HIN BNH.

1. t vn .Hin nay, ngnh thy sn ni chung v ngh nui tm ni ring ang pht trin rt nhanh, ng gp mt phn rt to ln vo s pht trin nn kinh t ca t nc. trc tip to ra cng n vic lm cho nhiu ngi dn ti cc khu vc c nui trng tm. Tuy nhin do vic pht trin t, t pht, thiu quy hoch c th to iu kin cho cc loi dch bnh bng pht v ly lan trn din rng ti cc a phng nui trng tm. Vi mc tiu pht hin v hn ch cc dch bnh trn tm, trong c bnh m trng, cc nh khoa hc nghin cu v tip cn n nhiu phng php chn on bnh m trng giai on tm con n tm trng thnh cho vic qun l bnh dch v nui trng c hiu qu, t b thit hi hn, Cc phng php chn on bao gm : PCR, ELISA,Western blot. Trong PCR c nh gi l c tnh ng dng cao nht trong cc phng php hin nay.

2. Tng quan v tnh hnh nui tm.2.1. Tnh hnh nui tm trn th gii.Ngh nui tm trn th gii xut hin cch y hng th k, nhng ngh nui tm cng nghip ch mi bt u t nhng thp nin 30 ca th k XX . Nm 1963 Motosaka-Fujinaga cng b cng trnh nghin cu v sn xut ging nhn to tm he (Nht Bn) v n nm 1964 quy trnh sn xut ging tm mi c hon chnh. T , ngh nui tm trn th gii mi pht trin mnh. Hin nay trn th gii c hai khu vc nui tm ln : th nht l cc nc ng nam Chu nh Trung Quc, Indonesia, Thi Lan, i Loan, n , Philipines, Vit Namcc nc ny chim 80% sn lng, th hai l cc nc Nam M gm Peru, Panama, Brazil,Nhng nm gn y tnh hnh dch bnh trn tm bng n v s suy thoi mi trng nui lm cho sn lng tm mt s nc gim mnh. Sn lng tm nui cng nghip trn th gii hng nm 258000 tn chim 36%, trn din tch nui 5%. Vi 5% din tch nhng sn lng 36% cho thy nui tm cng nghip t sn lng cao.2.2. Tnh hnh nui tm Vit Nam.Vit Nam l nc c kh hu nhit i v c b bin di 3200km rt thch hp cho ngh nui tm. Ngoi ra cn c nhiu ca sng ra bin to nn nhng vng nc l rt thun li cho vic pht trin thy sn ni chung v ngnh nui tm ni ring. Vit Nam c khong 500.000 ha t c kh nng nui tm, trong c khong 34.000 ha t nui tm bin. Din tch nui tm Vit Nam ngy cng tng ln do nhu cu tiu th trn th gii tng cng nh do gi c cao kch thch ngi dn m rng din tch nui tm. Din tch nui ngy cng tng nn sn lng cng tng theo. Vit Nam tn ti 3 hnh thc nui tm chnh : qung canh, bn thm canh v nui thm canh. Cng ngh nui tm nhng nm qua t c nhng tin b ng k. Tuy nhin, nhng nm gn y nhn chung nng sut nui tm ngy cng gim do bnh tt v mi trng nui . V th ngh nui tm cn c hng i ng n t c nng sut cao v n nh trong thi gian ti a ngh nui tm pht trin bn vng.

3. Tng quan v tnh hnh dch bnh trn tm.3.1. Trn th gii.Bnh m trng White spot disease (WSD) thng c bit n vi tn gi virus m trng- White spot syndrome virus ( WSSV) _ l mt mm bnh quan trng gy nh hng nghim trng i vi ngnh cng nghip nui tm trn ton th gii. Bnh m trng ln u tin c bo co t trang tri nui tm min Bc i Loan vo nm 1992 v Nht Bn nm 1993. Sau , bnh ny l nguyn nhn chnh gy nh hng rng khp ngnh cng nghip nui tm cc quc gia khc ti Chu v Chu M LaTin (bng 1). Ti Thi Lan, Bnh m trng ln u tin c bo co trn tm s ti cc tnh min ng v min Nam cc khu vc nui dc b bin ca Vnh Thi Lan v bin Andaman vo cui nm 1994. T l cht ca tm b nhim virus m trng ln ti 100% sau 3-10 ngy k t khi chng b nhim bnh.

Bng 1: Bng tng hp trnh t bng pht bnh m trng cc trang tri nui tm ti chu v chu M LaTin

Hnh 2: Tm s b bnh m trng.

Hnh 3: Tm th chn trng b bnh m trng.IHHNV c bit n l loi virus nh nht trn tm, l nguyn nhn gy t l cht nghim trng (>90%) trn tm ging v tm trng thnh P.Stylirostris, P.vannamei trang tri nui thm canh Hawaii. Sau khi c pht hin Hawii nm 1981 th IHHNV li c tm thy nhng tri nui tm M v tm t nhin b bin Thi Bnh Dng.Hin nay, ngy cng c nhiu loi virus xut hin trn tm. Theo Lightner (2003) th tm thy hn 10 loi virus gy bnh trn tm, ch yu l h tm he (Penaeid). L nguyn nhn gy t l cht cao v lm tht thot sn lng, nh hng nghim trng i vi ngh nui tm trn th gii.3.2 Vit Nam.Cng vi vic tng din tch, tng sn lng, tng mc thm canh th mi trng ngy cng b nhim dn n tnh hnh dch bnh cng tng nhiu hn. Nhng nm u thp nin 90, cng vi s pht trin ca ngh nui tm cng nghip, dch bnh tm cng bt u pht trin mnh m. Theo thng k ca b thy sn (1995) t nm 1993-1995 dch bnh tm bo ng trn ton quc lm thit hi hng t ng. Nm 2008 , din tch b thit hi l 658 ha ch yu l do bnh m trng. Tuy nhin, dch bnh tht s bng pht t nm 2010 n nm 2012 vi din tch thit hi ln n 7.068 ha, ch yu l do bnh hi chng hoi t cp tnh (B NN&PTNT 2013). Din tch nui tm b bnh tp trung ch yu vng ng Bng Sng Cu Long (BSCL) v mt s tnh khu vc Trung Trung B. BSCL l khu vc c phong tro nui tm pht trin mnh nht nc ta, t l sn lng nui tm chim hn 50% ca c nc. Trong thi gian t thng 12/2001 n nay, mt s tnh BSCL v ang xy ra dch bnh trn tc c cc m hnh nui, t l din tch nhim bnh khong 30-60% trn tng din tch nui (theo Khuyn ng Kin Giang, 2003) v c coi l i dch tm s. Trong , Tr Vinh, Sc Trng, Bc Liu v C Mau thit hi nng n nht. Cc vng nui c mn thp, t l mc bnh t hn so vi vng nui c mn cao. Cc thng nhit thp, ma ma, t l xut hin bnh thp hn cc thng ma kh, nhit cao. n nm 2013 tnh hnh dch bnh m trng v hi chng hoi t cp tnh gim i ng k so vi nm 2011 v 2012 (Tng cc thy sn 2013), nhng dn cn gy thit hi ln cho ngh nui. T ngy 01-06/7/2013 dch bnh trn tm nui nc l xy ra 6 tnh Ngh An, H Tnh, Ninh Thun, Tp H Ch Minh, Tin Giang v C Mau.Tng din tch nui b thit hi do bnh m trng l 18,43 ha, trong khi din tch b thit hi do hi chng gan ty cp l 27,97 ha. So vi tun trc, din tch nui tm b cht do 2 loi bnh trn thp hn l 279,02 ha. a phng c din tch b thit hi nhiu nht l tnh C Mau (16,72 ha), cc tnh cn li bnh ch xut hin ri rc v c c quan th y x l kp thi.Bng 4: Thit hi ca bnh tm m trng v hi chng gan ty cp cc tnh Vit NamDch bnhDin tch thit hi (ha)*a phng bo co dch bnh

WSSV18,43Ngh An, H Tnh, Ninh Thun, Tp H Ch Minh, Tin Giang v C Mau..

AHPNS27,97Ngh An, Ninh Thun, Tp H Ch Minh, v C Mau

* Ngun: Tng hp bo co tun t cc Chi cc Th y, Chi cc Nui trng thy sn v Chi cc Thy sn c giao nhim v qun l th y thy sn ca cc tnh ven bin.WSSW-bnh m trng; AHPNS-Hi chng hoi t gan ty cp

4. S lc v bnh m trng trn tm.4.1. Nguyn nhn bnh C 3 nguyn nhn gy nn bnh m trng tm l do virut, vi khun, v do mi trng.4.1.1. Mi trng.Khi mi trng nc nui tm c cng (Ca2+v Mg2+) cao, tm s hp thu qu nhiu Ca2+v Mg2+lm xut hin trn v nhng m trng. Tm trong trng hp ny c m trng v u ngc hoc phn v sng lng nhng vn kho mnh, khng c tm tp b, n tm vn hot ng v n u mc bnh thng nhng chu k lt xc di hn bnh thng v tm sinh trng hi chmBnh ny thng gp nhng mi trng nc c cng cao.4.1.2. Do vi khun (Bacterial White Spot Syndrome - BWSS).BWSS l vi khun c lin quan n mt s loi thuc h Bacillaceae, chng gy hi chng m trng tm.Trong giai on u nhim khun tm vn cn hot ng n mi v lt v, lc c th mt i cc m trng. Tuy nhin qu trnh lt v b chm li, chm ln v cht ri rc i vi tm b nhim nng nhng khng c hin tng tm cht hng lot, hu ht tm b ng rong, mang b bn. Tm bnh c cc m trng m c nhn thy trn v khp c th. Hin tng n mn lm lp v thoi ha v mt mu sc. m trng hnh trn, nh. Soi mu ti di knh hin vi m trng c dng lan ta hnh a y vi vin kiu g kha trn gia rng. Cc m trng thng ch pha ngoi lp biu b, tm c n chm hn nhng khng gy thit hi ng k.

Bnh m trng do vi khun c m t trn thng gp tm s nui Malaysia (Wang et al. 2000). Cc ao nui thm canh thng xut hin bnh m trng, nhng test PCR bnh WSSV m tnh.4.1.3. Do virut Gill associsted virus (GAV) v White Spot syndrome virus (WSSV)GAV l mt loi virut m nucleocapsidcos dng ng, v th virut hnh que, c v bao, hnh dng ging virut u vng. GAV c tm thy mch bch huyt v mang nhng cng c nhiu trong cc t bo mu. Khi b ly nhim cp tnh t bo mu s gim nhanh, cc c quan bch huyt b ri lon t chc.Dng lan truyn theo phng thc nm ngang m ch yu l n tht ln nhau, nhng vic lan truyn cng c th do ngun nc. GAV cng c th c truyn theo phng thc thng ng t b m cn khe, nhng by gi vn cha bit l vi-rut c trong trng hay khng.Tm b nhim bnh cp tnh thng l , km n, bi lu trn mt nc hoc quanh b. Thn tm thng bin thnh mu , c bit l cc ph b.GAV c pht hin u tin vo nm 1996, khi t l gy cht cao 4 nng tri Queensland, c, v c tm thy min nam x Walve v min Bc Territory vo nm 2004. WSSV l vi-rt dng hnh trng, kch thc 120 x 275nm, c mt ui ph mt u, kch thc 70 x 300 nm. Nhn c cu trc DNA dng vng vi 2 chui nucleotide v khng c th n (Occlusion body), b gen 292.967 bp. Vi-rt c t nht 5 lp protein vi trng lng phn t t 15-28 kilodalton. V bao c c ng knh khong 120-150 nm v chiu di 270-290 nm vi 2 lp protein VP28 v VP19, nucleocapsid c ng knh 65-70 nm, chiu di 300-350 nm vi 3 lp VP26, VP24, VP15.

Triu chng ca bnh l tm c rt nhiu m trng kch thc 0,5-2,0 mm xut hin bn trong v nht l gip u ngc (Hnh 1), t bng th 5, th 6 v lan ton thn. Bn cnh , tm bnh hot ng km, n nhiu t ngt sau b n, bi l mt nc hay dt vo b ao. i khi tm c du hiu thn. Bnh thng xut hin thi im 1-2 thng sau khi th nui, khi mi trng nui tm xu bnh d xut hin. Khi cc m trng xut hin sau 3-10 ngy tm cht hu ht trong ao nui (100%), t l cht cao v nhanh.WSSV ly truyn theo ng ngang l chnh, thng b ly t cc gip xc khc b nhim virut t bn ngoi hoc bn trong ao, hoc t cc loi chim nc n tm b nhim ri mang n ao, cng c th do chng n tm bnh cht trong ao nn t l ly lan v cng nhanh chng. Bnh ny khng th truyn theo chiu thng ng v cc non mang virut th khng chn nhng trong qu trnh sinh sn th tm c thi ra virut gy bnh nn u trng c th b bnh, nhng theo LO et al 1997 li cho rng n c th truyn thng.4.2. Phn b bnhBnh do vi rut WSSV c tm thy u tin i Loan- Trung Quc v Trung Quc lc a vo nm 1991-1992, Nht 1993 sau l n , Indonexia, Hn Quc, Malaysia, Thi Lan, Vit Nam. Ngoi cc nc chu cn c nhiu triu chng v m hc ca bnh m trng c thng bo M v Chu M, Latinh..4.3 .Phng v tr bnh.4.3.1. Phng nga bnh. - Th tm ng lch thi v khuyn co, khng th tm khi thi tit cha n nh, nht l nhit thp.- Mua con ging cc tri ging uy tn, c cht lng, tm b m c kim dch, tm Post c xt nghim m tnh vi bnh trc khi th nui.- Nu th nui nhiu ao, ngi nui nn chia thnh nhiu t khc nhau trnh nguy c xy ra dch bnh ng lot.- Trong qu trnh ci to ao cn dit ht cc vt ch trung gian l cc loi gip xc nh tm, cua.- Ao cn c ci to k, c bit l vt sch lp bn hi t v trc, cn c thi gian phi ao t nht t 7-10 ngy.- Nc cp vo ao cn c lc k qua li lc ngn trng c v gip xc lt vo ao.- Thc hin an ton sinh hc trong tri ging, trang tri nui tm nh thit lp hng ro cch ly bng li ngn chim v ng vt vo ao. Dng c s dng cho cc ao cn c st khun, khng s dng chung dng c cho nhiu ao.- Phng bnh trong qu trnh nui: Trong thi gian u v nui, tm cn nh sc khng yu. Bn cnh , thi im u v thi tit thay i nhiu. y l nguyn nhn lm cho tm d mn cm v pht bnh. c bit nhit nc v mi trng thp s to iu kin cho bnh pht trin. Trong qu trnh chm sc, nht l thng u tin, cn theo di cht ch cc yu t mi trng, kh c v x l kp thi. Trnh tm b stress.

4.3.2. Cha tr.-Trong giai on u ca bnh vic cha tr ch yu theo ng ming ca tm (qua thc n) v s ci thin v kh trng nc trong ao. Mt s loi thuc c n v khng c n nh thuc khng virus, khng khun, thuc bo v gan, mt v vitamin c dng kim sot bnh trong giai on ny. Sau thay nc v kh trng lm mt v sc sng ca mm bnh trong nc v ngn nga s ly nhim th cp. Tuy nhin phng php ny t c dng cha tr bnh trong giai on gia v giai on cui ca thi k pht bnh.-Cc loi thuc thng dng: Acid Chloric hoc acid oidic thng c s dng ci thin cht lng nc. Nhng loi thuc, ho cht chng virus thng gp l Iod. Cc loi ho dc v tho dc hay c s dng khng virus v ci thin h thng min dch cho tm l vitamin, cc nguyn t vi lng, aminoaxit...-Cch x l khi pht hin c du hiu nghi ng bnh m trng, mt vi con tp b nhanh chng vt ra khi ao. Dng SDKdit khun 1lt/1000m3 nc,Oxyxanhletomine1,5kg/1000m3 nc nh vo ao. Sau 2 gi nhTS 1001liu dng 2 lt/1000m3 nc +Bet-to-gane2 lt/1000m3 nc kt hp cho nTS 1001liu cao 5 ln/ngy, liu dng: 0,5 lt/10kg thc n, cho n ngy 3 c. chn ng virus m trng khng cho bng pht khp ao, tng cng sc khe cho tm bngVitamin C. X l mi trng bng cch dng TS B52 bui sng, bui chiu dngZeobt lng lc nc hm sau x l y bng men vi sinh Hatico.s liu cao gip vi sinh vt c li pht trin, gip tm khe nhanh hi phc. S kt hp trn s tng cng sc khng c th cho tm ng thi lm suy yu gim s pht trin virus. Phac iu tri bnh m trng cua Trng Sinh a c ap dung thanh cng t nhiu nm nay tai nhiu vung nui trn ca nc, c cac nha Khoa hoc, cac chuyn gia anh gia cao v hiu qua va khuyn khich s dung. 4.4. Phng php pht hin:4.4.1. PCR v Real- Time PCR.- K thut PCR.Du hiu nh bnh l pht hin sn phm khuch i ca on gen c hiu ca tc nhn gy bnh.- K thut Real-time PCR.Kthutny khngphn tch sn phm PCR trnbn thchnn khngcn sdngEthidiumbromide(lchtc cthgyt bin).Kthutny lmgimthigianbngthchvthigianchyin di.Thayvdngkthutin diphthinADNngi tathit kmt hthngquang hc ngaytrnmyPCRo cngnh sngsinh ratngngvi lngsn phm PCRckhuchi.4.4.2.Phng php M hc.-Mu c c nh 48 72h trong dung dch ha cht sau c c parafin, ct siu mng quan st t bo nhim di knh hin vi quang hc.-Hai phng php PCR v nh gi m hc c kh nng cho kt qu chnh xc, nhng mt nhiu thi gian v i hi phi c trang thit b tt, nn ch thch hp cc Trung tm nghin cu, cc Vin, Chi cc4.4.3.Que th nhanh.y l phng php mi, da trn nguyn l in di min dch, da trn c ch cc khng th n dng c kh nng bt ly cc protein v VP19 hoc VP28 ca WSSV. Phng php ny cho kt qu tc th, tin li cho ngi nng dn thc hin ngay ti ni mua ging, hoc kim tra ao nui khi thy c du hiu tm bnh. Mu tm c nghin trong mt ng nh, thm dung dch cn thit, ht dch nghin cho vo que th. Ch sau vi pht l c c kt qu da trn cc vch mu. Mc d tin dng, nhng gi thnh cn qu cao. Gi 01 mu th khong 300.000VN. Gn y VitNamcng nghin cu thnh cng que th nhanh, nhng gi vn cn cao. Ngoi ra phng php th ny ch c kh nng pht hin cc mu tm nhim lng virus WSSV ln, nhy khng bng phng php PCR hay nhum ha m min dch.4.4.4. Phng php lai DNA ti ch (In situ DNA hybridization).Du hiu nh bnh l kt qu lai gia nhng on gen dng lm mu d v on gen ca tc nhn gy bnh trong iu kin nghim ngt.4.4.5. Mt s phng php khc.Ngoi cc phng php ni trn, pht hin bnh m trng trn tm, mt s phng php khc cn c s dng nh ELISA, Western blot, Southern blotTrong s cc phng php trn, nu so v nhy, thi gian tin hnh, yu cu trang thit b v gi thnh th cc phng php PCR ni chung c cc c im ph hp nht, c kh nng p ng c cc yu cu v pht hin bnh trn tm.

CHNG 2. TNG QUAN PCR

1. S lc v PCR.PCR (Polymerase Chain Reaction) l mt k thut da trn c s ca phn ng ko di primer nh enzyme Taq polymerase khuch i in vitro cc nucleic acid c hiu trong my lun nhit hay cn gi l my PCR.PCR cho php khuch i theo hm m n hng triu ln cc on DNA c chiu di t 200 3000 bp. on DNA c khuch i c nhn dng nh cp primer c hiu l mt oligonucleotide thng c thit k vi chiu di khong 20 nucleotide.2. Nguyn tc.Enzyme DNA polymerase c dng trong phn ng PCR l Taq Polymerase (gi tt l Taq pol), mt enzyme chu nhit c ly vi khun sng trong iu kin nhit cao Thermus quaticus. Ngoi Taq polymerase th phn ng PCR cn cn c bn loi deoxyribonucleotide (dATP, dCTP, dGTP, dTTP) v hai primer trn c s khun mu ca mt on DNA nht nh hoc cha bit trnh t.Cc on DNA mi hnh thnh li c s dng lm khun mu. Sau nhiu chu k, s lng on DNA ni trn c nhn ln gp nhiu ln, khi c th s lng cn thit s dng cho vic phn tch, to dngHai promer trong PCR mt primer s tc ng ln si DNA chiu 3 5 gi l primer thun (Forward Primer). Primer cn li tc ng theo chiu 5 3 gi l primer ngc (Reverse Primer).

Hnh: S phn ng PCR

Nh s trn, t chu k th hai v sau, Taq pol bt u to ra cc on DNA c chiu di xc nh. PCR thng tin hnh khong 25 35 chu k, qua t 10-6 g DNA ban u c th khuch i (amplification) ln ti 1 g (khong 2kb). Mi chu k PCR bao gm ba giai on c nhit khc nhau.Gy bin tnh (denaturation) 90 950C: Giai on ny, phn t DNA ang dng xon kp s b tch ra thnh hai s n. Tt c cc phn ng enzyme trong giai on ny u b dng li.Gn mi (annealing) 40 650C: Giai on ny cc primer gn vo cc v tr c trnh t tng ng DNA khun. Khi cc primer lp rp chnh xc, Taq pol c th bt u qu trnh sao chp khun mu. Trong giai on ny, phm vi nhit s dng c th rt rng v ty thuc vo trnh t nucleotide ca primer. Thng thng khong 550C nhng i khi ch 350C hoc ln n 680C.Ko di phn t (extension) 70 720C: y l khong nhit ti u cho Taq pol tin hnh tng hp DNA bng cch b sung cc dNTP bt u t cc v tr c primer theo chiu 5 3.

Hnh: Cc chu k ca phn ng PCR3. Cc loi PCRc pht minh bi Kary Mullis vo nm 1985, cho n nay, k thut PCR c rt nhiu bin i v pht trin thnh cc kiu khc nhau da trn phng php PCR c in (Conventional PCR) ban u ph hp vi mc ch s dng. Nhng nhn chung v c bn cc k thut PCR hin nay u hot ng trn cng mt nguyn tc nh nhau v s khc bit ln nht ch nm giai on thu nhn kt qu ca phn ng PCR. V vy theo mt cch nhn ch quan ca nhm, k thut PCR c th c phn loi c bn da trn phng php thu nhn kt qu sau phn ng. Theo , cc phn ng PCR c kt qu c thu nhn vo thi im cui cng v c c thng qua phng php in di trn gel c gi chung l end-point PCR. Ngc li, cc k thut PCR c kt qu c thu nhn trong sut gian on tin trnh phn ng xy ra c gi chung l Real time PCR.

3.1. End-point PCR.i vi cc phng php PCR thu nhn kt qu thng qua chy in di trn gel ny, yu cu thc hin phn ng PCR gn nh tng t nhau, mt phn ng s c thit lp khi c y primer, DNA bn gc (hoc RNA), dNTP, Taq pol v my lun nhin thng thng. Cc phng php ny ch khc nhau mt tng i nh cc bc tin hnh hay cc thit k cc primer m thi. V d mt s phng php PCR c bn trong nhm cc phng php End-point PCR hin nay.PCR c in: Mc d k thut ny ra i rt lu, tuy nhin n nay vn cn c s dng ph bin v tnh n gin trong thao tc, khng yu cu cao v iu kin thit b. Trong k thut ny, DNA khun mu c tho xon kp sau hai primer tc ng hai u si n v DNA mi c tng hp nh Taq pol vi bn loi deoxyribonucleotide.

Hnh: S PCR c in.PCR m neo (Anchored PCR): K thut ny yu cu ch cn bit trnh t nucleotide mt u ca khun mu v gn mt ui ng trng hp nhn to vo u kia (v d dA). Sau DNA c khuch i nhiu ln nh mt primer c trnh t bit trc v mt primer th hai c oligo (dT).

Hnh: PCR m neo

PCR o ngc (inverse PCR): c dng khuch i cc phn t k cn vi on c trnh t DNA bit trc, phn t DNA ny c ct hn ch v ni li nh enzyme DNA ligase to thnh mt vng trn c tnh cht monomer, sau DNA c khuch i nh hai primer tng ng vi u ca trnh t bit trc.

Hnh: PCR o ngc.PCR phin m ngc (Reverse transcription PCR): RT-PCR l phng php PCR xc nh s hin din acide nucleic ch l RNA. c th thc hin c k thut PCR ny th trc ht RNA cn c phin m ngc thnh Complementary DNA (cDNA). Sau s dng k thut PCR c in thng thng khuch i trnh t ch trong cDNA bng cp mi c hiu cho trnh t ny. Trong k thut ny, giai on phin m ngc rt quan trng v mang tnh quyt nh s thnh cng ca phn ng RT-PCR. Enzyme c s dng trong giai on ny l Reverse transcriptase c tch chit t Moloner murine leukemia virus (M-MLV), mt loi virus gy bnh ung th bch cu trn chut hoc t Avian myeloblastosis virus (AMV) gy bnh ung th ty trn gia cm. Enzyme ny l mt loi DNA polymerase khng chu nhit, s dng RNA mch n lm khun tng hp ra DNA. enzyme Reverse transcription ny c th tng hp ra cDNA t RNA cng cn phi c mi c hiu gn vo RNA khun v c s hin din ca dNTP ko di cDNA.

Hnh: RT-PCR

Nested PCR: K thut ny l mt dng thay i ca PCR c in, trong c hai cp mi PCR c dng khuch i on DNA qua hai giai on phn ng khc nhau. Lu u, phn ng PCR c thc hin trong 15 n 30 chu k vi cp mi th nht khuch i on DNA di hn c cha on gen cn xc nh. Sau sn phm PCR ln th nht s c lm khun thc hin tip phn ng PCR ln th hai vi cp mi th hai v khuch i on gen cn xc nh. Trong k thut ny, tnh c hiu ca primer c nng ln s gim vic hnh thnh cc sn phm khng mong mun xung mc thp nht. Ban u k thut ny phi c s hn ch do d b nhim v phi m tube PCR chuyn mu thc hin phn ng ln th hai. Tuy nhin ngy nay vi s tin b trong k thut thit k cc primer nn cho ra i cc b kit thc hin Nested PCR mt ln khng cn phi m tube PCR nn kh nng tp nhim c gim xung rt thp.

Hnh: Nested PCR

3.2. Real time PCR.V nguyn tc, sn phm to ra trong real time PCR cng tng t nh trn cc phn ng thuc nhm end-point PCR ni trn. Tuy nhin so vi cc phng php , Real time PCR c th c xem l s tin b trong phn ng PCR v qu trnh lm phn ng nhanh, nhy cao, khng cn phi thc hin cc phn ng sau khi PCR c kt qu. Tuy nhin thc hin c Real time PCR iu cn thit nht l phi c my Real time PCR. My ny cng c mt bung nhit chy c chng trnh lun nhit nh my PCR bnh thng nhng c thm mt thit b quang hc c hai chc nng l pht ra ngun sng kch thch c bc sng xc nh ln cc tube PCR ang phn ng v mt camera hay cm bin quang hc c kh nng ghi nhn c nh sng hunh quang pht ra t cc tube PCR ang phn ng c chiu tia sng kch thch.

Hnh: S mt thit b real time dng n tungsten lm ngun sng kch thch v CCD camera ghi nhn ton b tn hiu hunh quang ca cc tube phn ng c chiu ngun sng kch thch.

Thit b real time dng si quang hc a nh sng kch thch n tube phn ng.

H thng 6 n LED cng vi 6 cm bin quang c gn trn mt gi v c di chuyn st trn bung nhit c trong thit b real time.

Trong real time PCR, cht hunh quang cng quan trng khng km my real time PCR. V nguyn tc, cht hunh quang ny phi lm th no tube PCR ang phn ng c th pht c hunh quang khi b chiu ngun sng kch thch vo lc trong tube PCR c s hin din ca cc sn phm PCR t DNA ch. Ngc li s khng pht hunh quang c nu khng c sn phm khuch i trong tube PCR. Cho n nay c rt nhiu cht hunh quang c kh nng nh vy c tm thy. Nhng nhn chung, cc cht hunh quang c bit n vi hai dng:Th nht l cht pht hunh quang l mt loi mu hunh quang chn vo s DNA mch i. Nguyn tc ca k thut real time PCR dng mu hunh quang chn l khi khng c s hin din ca sn phm khuch i, cht hunh quang b phn tn trong dung dch PCR mix v tube phn ng v vy s khng pht hunh quang hoc pht hunh quang vi tn hiu rt yu. Nhng khi sn phm khuch i ca PCR xut hin, mu hunh quang s tp trung chn vo cc s DNA mch i v cng pht hunh quang ca cc tube PCR s tng dn theo lng sn phm khuch i c to ra. Ethidium bromide l cht c s dng lm mu hunh quang ban u, tuy nhiu cht ny c thay th bng SYBR I vi cc u im vt tri hn nh mc mu hunh quang nn thp, kh nng chn vo mch DNA i cao hn, khng lm hai mch DNA gn qu cht vo nhau khin kh tch ra khi gy bin tnh DNA.Th hai l real time PCR s dng probe lm cht pht hunh quang. y, probe c tm dch l on d. y l nhng oligonucleotide mch n c trnh t bt cp b sung vi mt trnh t t bit trn DNA khun. S dng probe lm cht pht hunh quang da trn nguyn tc l khi c mt sn phm khuch i c hiu trong ng phn ng th s c s bt cp ca probe ln trnh t c hiu ca sn phm khuch i, khi c s bt cp ny th s c s pht hunh quang t ng phn ng khi c chiu ngun sng kch thch. C nhiu loi probe khc nhau, i khi primer cng c s dng lm cht pht hunh quang trong real time PCR. Taqman probe l mt trong s cc loi probe c dng ph bin trong cc phn ng real time PCR hin nay. V nguyn tc ca k thut ny, ngoi cc thnh phn c bn, real time PCR mix cn cha thm hai thnh phn c bn tube PCR c th pht hunh quang c l Taq probe v enzyme Taq polymerase c hot tnh 5 3 exonuclease ct b probe khi probe ny bt cp ln si khun v cn u 3 ca primer khi enzyme ko di primer tng hp mch b sung. Taqman probe l mt on nucleotide ngn, u 5 c gn mt cht pht hunh quang gi l reporter. u 3 cn li c gn mt cht c kh nng hp th c nh sng hunh quang do reporter pht ra c gi l quencher. C hai loi quencher l quencher pht hunh quang v quencher pht nhit. i vi quencher pht hunh quang, khi reporter pht ra hunh quang, quencher s hp th nh sng hunh quang t reporter v chuyn nng lng hp ph c thnh nh sng. V vy hunh quang ca reporter s khng n c cm bin c hunh quang mc d c nh sng ca quencher pht ra do knh lc pha trc cm bin hunh quang ch cho nh sng t reporter i qua. i vi quencher pht nhit, ton b hunh quang do reporter pht ra s c hp ph v chuyn thnh nhit.C ch pht hunh quang ca Taqman probe nh sau. Khi cha c s xut hin ca sn phm khuch i c bit t DNA khun th Taqman probe vn cn nguyn vn do vy hunh quang c pht ra t reporter b quencher hp ph, tube PCR s khng pht c hunh quang. Khi bt u c s xut hin sn phm khuch i c hiu th Taqman probe s bt cp vo DNA khun ca sn phm ny nhit bt cp v s b enzyme Taq polymerase ct b ko di mch tng hp. V vy cc quencher s b ct ri khi Taqman probe v hunh quang t reporter s n c cm bin.

Hnh: c ch hot ng ca Taqman probe

CHNG 3. CHI TIT MT S PHNG PHP PCR DNG TRONG CHN ON BNH M TRNG TRN TM.

Hin nay, phng php PCR c dng rt ph bin trong chn on pht hin WSSV trn tm, tt c cc phng php ny u xoay quanh hai mc tiu l nh tnh WSSV hoc nh lng WSSV. nh tnh WSSV ngi ta c th dng PCR c in hoc Nested-PCR, hai phng php ny u c im chung l phi chy in di trn gel sau khi PCR c kt qu. Tuy nhin khi xt v nhy v tnh c hiu khi lm PCR th phng php Nested-PCR vt tri hn PCR c in. V vy Nested-PCR c th c xem l bin php ti u hin nay v gi thnh, nhy, yu cu trang thit b thc hin vic nh tnh WSSV. nh lng WSSV ngi ta dng Realtime-PCR, ngoi vic nh lng c WSSV, phng php ny cn c th nh tnh c WSSV, nhy ca phng php ny l rt cao nu so snh vi Nested-PCR, tuy nhin do nhy qu cao nn phn ng rt d b tp nhim khi tin hnh. Bn cnh , thc hin c phn ng Realtime-PCR cn phi c my Realtime-PCR v gi thnh ca phn ng Realtime-PCR hi cao hn so vi Nested-PCR. V vy Realtime-PCR khng c dng ph bin nh Nested-PCR.

1. Realtime-PCR trong pht hin v nh lng WSSV[1].1.1. Ha cht dng c.Mu xt nghim: Mu tm ging: mang, chn b, chn bi ca tm nui thng phm v tm b m.Dng c: H thng Real-time PCR, t cy v trng, t lnh lu mu, my khuy t, b iu nhit, my ly tm, My vortex, Micropipet (0.5 n 1000l), cn phn tch, bp in, b in di, dng c nghin mu dng cho eppendorf (50 ng chy nha, 50 eppendorf 1,5ml).Ha cht: B thuc th ly trch kh DNA tm: nc ct (50ml), NaOH (350l), SDS (150l), 48l hn hp Real-time PCR gm: primer, dNTP, Taq polymerase, TaqMan probe, Mg2+, dung dch m PCR - (tt c x50 eppendorf). C th nh sau cho mi phn ng:

RT-PCR Pre-Mix reagent 7.0l

Iqzyme DNA Polymerase 2UI/l0.5l

Reverse Transcriptase (RT) Enzyme Mix0.5l

Total: 8.0l

i chng m (control (-)): 100l dung dch TE 1X. i chng dng (control (+)): 100l DNA ca WSSV. 5 chun (standard (std)): 50l mi chun cha DNA ca WSSV vi hm lng ln lt l: 105 bn sao/2l, 104 bn sao/2l, 103 bn sao/2l, 102 bn sao/2l, 101 bn sao/2l

1.2. Phng php tin hnh.1.2.1. Thu mu v bo qun:Mu tm ging: ly mu nguyn 50-70 con. Ty s lng tm trong n nhng tng trng lng mu khng qu 1g. Mu tm thu cn sng hay c c nh trong cn 95o.Mu tm thng phm: ly mt phn nh mang, chn bi, chn b ca 10-15 con nhng tng trng lng mu khng qu 1g. Mu tm thu cn sng hay c c nh trong cn 95o.Mu tm b m: thu mu tm b m phc tp hn v phi m bo tm vn cn sng tham gia sinh sn. Do vy u tin thu mu chn bi 2 hoc phn mang nhng tng trng lng mu khng qu 1g. Mu tm thu cn sng hay c c nh trong cn 95o.Cc mu tm thu ti ni ly mu phi c nh trong cn 95o theo t l tm:cn=1:3. Mu sau khi c nh trong cn c th c lu nhit thng. Khi bo qun lu hn phi thay cn mi v bo qun trong t -20oC.1.2.2. Pha dung dch ly trch mu: Dng micropipet ht 125 l NaOH vo l ch 50ml nc ct, lc u. Ht 62.5l SDS cho tip vo l cha nc ct ny, lc trn u.Dung dch ny c bo qun trong bnh ti, c th lu trong 3 thng.1.2.3. Tin hnh ly trch: Cho mu tm vo ng eppendorf 1.5ml, thm 100l dd tch chit, dung chy nh nghin nhuyn. Sau cho thm 500-700l dd tch chit, tip tc nghin. Chuyn cc ng sang b n nhit 96oC, trong 5-10 pht. Lm lnh nhanh. Ly tm 13.000 vng/pht trong 5 pht. S dng dch ni (c th bq -20oC). Ht 20l phn dch ni sau khi ly trch bng SDS, NaOH cho vo eppendorf 1.5ml. Sau thm 200l dd Instagene Matrix ( khuy sn) vo trong ng. Tin hnh nhit 56oC trong vng 15-30, ly ra vortex 10 giy. Tip tc 100oC trong 8-10, vortex trong 10 giy. Ly tm 13.000 vng/pht trong 3 pht. S dng dch ni (c th bq -20oC)1.2.4. Tin hnh Real-time PCR: Cho 2l dch ly trch DNA cn phn tch vo cc eppendorf c cha sn 48l hn hp Real-time PCR t th tch phn ng l 50l. i chng m: cho 2l i chng m vo eppendorf c cha 48l hn hp Real-time PCR t th tch phn ng l 50l. i chng dng: cho 2l i chng dng vo eppendorf c cha 48l hn hp Real-time PCR t th tch phn ng l 50l. i vi chun: s dng 5 chun c bn sao WSSV vi hm lng ln lt l: 105 bn sao/2l, 104 bn sao/2l, 103 bn sao/2l, 102 bn sao/2l, 101 bn sao/2l, cho 2l chun WSSV vo eppendorf c cha 48l hn hp Real-time PCR t th tch phn ng l 50l. Thit lp qu trnh lun nhit:

Chu k 1 - lp li 1 lnChu k 2 - lp li 45 ln

Bc 1: 95oC trong 330sBc 1: 95oC trong 45sBc 2: 60oC trong 1

Thit lp v tr t mu: mu cn xt nghim (unknown), chun (standard), i chng (+) v (-). Sau chn v tr t mu trn bung a 96 ging qua mn hnh my tnh. nh tn mu: mu c nh tn tng ng vi s th t mu gc hay ngun gc mu thun tin cho phn tch kt qu. Chn cht ch th mu hunh quang: chn FAM v mu hin th cho FAM hoc dng cht khuch i in di trn gel agarose 1% ch Ethydium Bromine kim tra.1.2.5. Phn tch kt qu Real-time PCR:Kt qu sau khi thc hin Real-time PCR c x l trn phn mm v c phn tch qua th biu din c mi quan h nng hunh quang v s chu k, th biu din ng chun, bng kt qu chy mu. th biu din mi quan h gia nng hunh quang v s chu k:Trong thi gian chy PCR, ta quan st thy c biu hin ng thi nng DNA ca WSSV, mi quan h hunh quang v s chu k phn bit m tnh, dng tnh.

th biu din mi quan h gia nng hunh quang v s chu k biu din qua dng log (log view) n v hunh quang trc Y c tnh dng log. Ta quan st thy: th biu din mu dng tnh c ng biu din tnh hiu hunh quang vt qua ng Baseline subtracted (ng tr i tn hiu nn). th biu din mu m tnh c ng biu din tnh hiu hunh quang nm di ng Baseline subtracted (ng tr i tn hiu nn).Trong hnh xc nh c chu k ngng (khi ng base line ct tt c cc ng biu din nng hunh quang ca mu kim tra). Sau ta tin hnh phn tch ng chun v bng kt qu bit c hm lng WSSV c ban u 2l trong mu th. th biu din ng chun:

Ta thy trn hnh c: trc tung (Chu k ngng) v trc honh (gi tr logarite s lng bn sao ban u c n v l s bn sao). T ta xc nh ng phng trnh Y=a.X+b. th ny th hin mu dng tnh unknown qua chu k ngng v s bn sao, s bn sao trnh t ch cng nhiu tng ng vi s chu k ngng cng thp.T th ng chun ta cn xc nh c h s tng quan (R2), hiu qu phn ng v nghing (slope).H s tng quan: cng cao tng ng gi tr cng chnh xc. HSTQ c gi tr gia [0;1] da theo bin thin t gi tr tng ng rt km chnh xc cho n hon ho.Hiu qu phn ng v nghing: 2 i lng ny c s tng quan cht ch vi nhau. Hiu qu phn ng cho bit thng tin v ha cht phn ng. nghing ca ng chun tng quan hiu qu phn ng ( nghing -3.332 cho hiu qu 100% phn ng RT-PCR, t suy ra cao hay thp hn 100%).Hiu qu = [10(-1/slope)] 1 (slope: nghing)

Bng kt qu chy mu: c xut ra t my:WellTypeIndentifierRepCtLog SQSQSQ MeanSQ SDCt MeanCt SD

A01

A03

A05

A07

A09

Well: V tr ging Type: Loi mu Indentifier: nh tn mu Rep: v tr mu trn 96 ging Ct: Chu k ngng SQ: s bn sao ban u SQ: lch chun Mean: trung bnh

Kt qu kim tra mu qua Real-time PCR s c phn tch ch yu da trn: s bn sao ban u (SQ), gi tr chu k ngng (Ct). Khi mu b lp li, kt qu s xut hin bn sao trung bnh (SQ Mean), gi tr chu k ngng trung bnh (Ct Mean), lch chun bn sao ban u (SQ SD), lch chun ca chu k ngng (Ct SD), v tr t mu, loi mu gip ngi phn tch bit r thng tin cn kim tra.2. Nested-PCR trong pht hin v nh lng WSSV[2].2.1. Ha cht dng c.Mu xt nghim: Mu tm thu thp trong ao nui cng nghip c thu gi v bo qun trong cn 95o.Dng c: H thng Non-stop Nested PCR, t cy v trng, t lnh lu mu, my khuy t, b iu nhit, my ly tm, My vortex, Micropipet (0.5 n 1000l), cn phn tch, bp in, b in di, dng c nghin mu dung cho eppendorf (50 ng chy nha, 50 eppendorf 1,5ml).Ha cht:- Ha cht ly trch DNA: NaOH, SDS, TE 10X, Ethanol 70 %. - Ha cht PCR: Taq DNA polymerase, PCR buffer 10X, MgCl2 25 mM, dNTP 25 mM, mi, nc ct. - Ha cht in di: Agarose, Ethiumbromide 10 mg/ml, Loading dye, thang DNA 1000 bp, dung dch m TBE 10X. - B kt tinh sch DNA (Wizard SV Gel and PCR Clean - Up System).- i chng m (control (-)): 100l dung dch TE 1X.- i chng dng (control (+)): 100l DNA ca WSSV.Trnh t on mi c s dng trong nghin cuTn miTrnh tKch thc sn phm

Mi 146F15-ACT-ACT-AAC-TTC-AGC-CTA-TCTAG-31447 bp

Mi 146R15-ACT-ACT-AAC-TTC-AGC-CTA-TCTAG-3

Mi 146F25-GTA-ACT-GCC-CCT-TCC-ATC-TCC-A-3941 bp

Mi 146F25-TAC-GGC-AGC-TGC-TGC-ACC-TTG-T-3

2.2. Phng php tin hnh.2.2.1. Thu mu v bo qun:Tm ly mu nguyn 50-70 con. Ty s lng tm trong n nhng tng trng lng mu khng qu 1g. Mu tm thu cn sng hay c c nh trong cn 95o.Cc mu tm thu ti ni ly mu phi c nh trong cn 95o theo t l tm:cn=1:3. Mu sau khi c nh trong cn c th c lu nhit thng. Khi bo qun lu hn phi thay cn mi v bo qun trong t -20oC.2.2.2. Pha dung dch ly trch mu:- Dng micropipet ht 125 l NaOH vo l ch 50ml nc ct, lc u. - Ht 62.5l SDS cho tip vo l cha nc ct ny, lc trn u. Dung dch ny c bo qun trong bnh ti, c th lu trong 3 thng.2.2.3. Tin hnh ly trch:- Cho mu tm vo ng eppendorf 1.5ml, thm 100l dd tch chit, dng chy nh nghin nhuyn. Sau cho thm 500-700l dd tch chit, tip tc nghin.- Chuyn cc ng sang b n nhit 96oC, trong 5-10 pht. Lm lnh nhanh.- Ly tm 13.000 vng/pht trong 5 pht. S dng dch ni (c th bq -20oC)- Ht 20l phn dch ni sau khi ly trch bng SDS, NaOH cho vo eppendorf 1.5ml. Sau thm 200l dd Instagene Matrix ( khuy sn) vo trong ng.- Tin hnh nhit 56oC trong vng 15-30, ly ra vortex 10 giy.- Tip tc 100oC trong 8-10, vortex trong 10 giy.- Ly tm 13.000 vng/pht trong 3 pht. S dng dch ni (c th bq -20oC)2.2.4. Chun b hn hp thc hin phn ng Non-stop Nested PCR- Dng micropipet ht 2l dung dch tch chit DNA ca mu cn phn tch cho vo ng eppendorf cha sn 48l hn hp Non-stop Nested PCR t th tch 50l.- i chng m: cho 2l i chng m vo eppendorf c cha 48l hn hp Non-stop Nested PCR t th tch phn ng l 50l.- i chng dng: cho 2l i chng dng vo eppendorf c cha 48l hn hp Non-stop Nested PCR t th tch phn ng l 50l.2.2.5. Thit lp chng trnh thc hin Non-stop Nested PCR:Thit lp qu trnh lun nhit:

Chy k 1Lp li 1 ln40oC trong 10

Chy k 2Lp li 1 ln95oC trong 5

Chy k 3Lp li 20 lnBc 1: 94oC trong 30s

Bc 2: 58oC trong 30s

Bc 3: 72oC trong 1

Chu k 4Lp li 40 lnBc 1: 94oC trong 30s

Bc 2: 51oC trong 30s

Bc 3: 72oC trong 1

Chu k 5Lp li mt ln72oC trong 7

Gi 4oC cho n khi phn tch

Tin hnh chy Non-stop Nested PCR theo thng s trn my

2.2.6. in di mu.- Chun b gel agarose 2%: cho 4gram agarose vo 200ml dung dch TBE 0,5X, un chy hon ton, ngui khong 60oC ri thm 10l ethydium bromine (10mg/ml), trn u, khun, gel ngui khong 30. Sau a vo khay in di cha dung dch TBE 0,5X. - Np (load) mu: chun b mu th, cho 12l dung dch np mu (loading buffer) (c glycerol v bromophenol blue) vo trong mi mu th. Dng micropipette cho 15l mu v 15l thang DNA chun vo ging.- in di gel agarose: in di trong thi gian 30 100V v 50mA.2.2.7. Phn tch kt qu Nested PCR.Th mt vi v d bn in di sau khi chy Non-stop Nested PCR:- Ta thy c hiu cho thy quy trnh c c hiu cao vi WSSV v ch to sn phm khuch i vi mu tm nhim WSSV (3), vi mu IHHNV (4) th khng c.

Kt qu in di c hiu ca quy trnh. Ging 1: thang 1000 bp; ging 2: i chng dng; ging 3: mu tm nhim WSSV; Ging 4: mu tm nhim IHHNV; ging 5: i chng m.- Kt qu xc nh nhy cho thy nhy ca quy trnh nested PCR t 103 bn sao. Ngi ta xc nh c nhy ca quy trnh nesded PCR l 102 bn sao khi tin hnh trn chun WSSV DNA . Nguyn nhn ca s khc bit c th l do cc nghin cu s dng Taq polymerase c hot tnh khc nhau.

nhy ca quy trnh nested PCR pht hin WSSV v i chun gc WSSV -DNA. Ging 2, 3, 4, 5, 6, 7, 8, 9, 10 tng ng vi s bn sao/l 108, 107,106,105, 104, 103, 102, 101, 1; ging 11: i chng m, ging 1: thang DNA 1000 bp.3. Mt s gen ca WSSV c khuch i trong phn ng PCR. mc 1 v 2 ca chng 3, phng php c th ca Realtime PCR v Nested - PCR c nu ra mt cch c th. Tuy nhin hai phn ng PCR trn u dng cc b kit PCR thng phm v trnh t on gen c khuch i nhn bin WSSV khng c nu ln c th v l do b mt cng ngh. Tuy nhin vi mt s b kit ph bin, cc on gen c dng khuch i trong phn ng PCR nhn bit WSSV thng l cc on gen in hnh sau[3]:- ORF126/ie1:

- VP28:

- ORF24/ie2:

4. Ga mt s loi kit PCR chn on WSSV trn tm c bn trn th trng Vit Nam hin nay.Trn th trng hin nay, phn ln cc loi kit thng mi trn th trng l ca nc ngoi do c gi c cnh tranh v cht lng. Cc cng ty Vit Nam u t vo lnh vc sn xut kit PCR chn on WSSV hin nay cha nhiu v mnh bng cc cng ty nc ngoi. in hnh trong s cc sn phm ang lu hnh trn th trng hin nay l cc b kit PCR ca nh sn xut Farming IntelliGene - i Loan, gi thnh cho mt b kit Nested PCR l 1.450.000VN, b kit Realtime PCR l 1.650.000VN, mi b kit c th thc hin 200 phn ng. Vi mc gi hin ti, cc b kit PCR ny c xem l c gi c rt cnh tranh. Tuy nhin gi thnh thc hin mt th nghim kim tra tm c nhim WSSV hay khng hin nay ti cc phng th nghim a phng vo khong 800.000 1.200.000 VN, vi mc gi ny i vi ngi nng dn c nhu cu xt nghim tm nhim WSSV vn cn cao. Nguyn nhn ca vic gi thnh cho mi ln kim tra tng ln cao n mc nh vy l do khi thc hin PCR, ngoi b kit c gi tng i thp th tt c cc ha cht t khu bo qun, x l mu, tch chit DNA t tm u phi nhp khu l chnh.5. Mt s u xut cho cng tc chn on WSSV trn tm hin nay.Li ch v chnh xc ca k thut Nested PCR hay Realtime PCR hin nay l khng th ph nhn c. Tuy nhin phng php PCR hin nay thng mt khong 2 ngy t lc nhn mu n lc tr kt qu. Thng vi thi gian nh vy s gy bt li cho ngi dn trong khu kim tra trc khi mua tm ging hoc chn on bnh cho tm ca h. V vy phng php chn on WSSV bng que th nhanh mi ra i trong thi gian gn y c th c xem nh l mt gii php mi c kh nng rt ngn thi gian kim tra WSSV. Tuy nhin k thut ny hin nay vn cn gp mt hn ch l s lng copies ca virus phi cao ( nhy cn km) vn cn phi c nghin cu thm. Trong khi vi phng php Nested PCR hoc Realtime PCR ch cn lng copies ca virus t 20 200 l c th pht hin c mt cch d dng. Do nn bin i la chn linh hot khi quyt nh dng phng php no cho ph hp khi mun kim tra WSSV cho tm.

Mt khc, cn phi thc y vic nghin cu ni a ha cc ha cht trong th nghim kim tra WSSV gp phn gim gi thnh cho mi ln kim tra xung thp hn na to iu kin tt hn cho ngi dn. Nu trong nht thi, vic u t cc my Realtime PCR cha kh nng th vic la chn Nested PCR c th chy trn cc my PCR thng c xem l mt la chn ti u cho hin ti. Tuy nhin v lu di th nn trang b cc h thng Realtime PCR l tt nht.

Ti liu tham kho:

1.Cng ty Nam Khoa, .h.N.L.T.H., Pht hin v nh lng virus gy bnh m trng (wssv) trn tm s (penaeus monodon) bng k thut realtime-pcr. 2005.2.Hunh ng Sang, T.T.K.K., H Th Bch Trm, Phm Th Thu Hng, Hunh Thanh Trc, L nh n, Nghin cu pht hin vi-rt m trng (wssv) trn tm ti tnh c mau bng nested pcr.3.Kou, G.H., C.F. Lo, and W.J. Liu, Promoter sequences from WSSV immediate early genes and their uses in recombinant DNA techniques. 2008, Google Patents.

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