B GIO DC V O TO TRNG I HC NNG LM TP.

H CH MINH VIN NGHIN CU CNG NGH SINH HC V MI TRNG

TI LIU HNG DN THC HNH CHN ON VI-RT M TRNG TRN TM BNG K THUT NESTED - PCR

Cn b hng dn Hunh ng Sang

Thng 9 nm 2012
Lu hnh ni b

Bi 1. BNH M TRNG
1. Tnh hnh dch bnh m trng ti khu vc ng bng sng Cu Long trong nhng nm gn y Theo Chi cc Thy sn Tin Giang, nm 2010, s lng thit hi bnh m trng trn tm, gy thit hi 66.972.000 con/275,82 ha, chim t l 8,3 % lng ging th nui v 12,1 % din tch th nui. Nm 2011, din tch tm nui b thit hi l 951,04 ha, chim t l 18,3% din tch th nui. Nguyn nhn chnh l do hoi t gan ty (chim khong 70% din tch thit hi), cn li l do bnh m trng. Ti tnh C Mau, nm 2011, din tch tm nui cng nghip b bnh 538,85 ha (m trng 86,33 ha, hoi t gan ty 452,53 ha). Trong 4 thng u nm 2012, ton tnh c 555 ha (c tm s v tm th chn trng) b cht, tp trung nhiu cc huyn m Di (vi 234 ha tm cht), Ph Tn (215 ha), Ci Nc (67 ha) v thnh ph C Mau (38 ha). Tm cht ch yu bi cc bnh m trng, hoi t gan ty Trn a bn tnh Tr Vinh c 3.351 ha tm nui, vi gn 300 triu con tm s b cht ti cc huyn Tr C, Cu Ngang, Duyn Hi, Chu Thnh, c thit hi hn 300 t ng. S din tch ny nm trong tng din tch 19.323 ha, vi hn 1,5 t con tm s th nui u v n nay ti cc huyn trn. Tm cht trong giai on t 15 45 ngy tui, ch yu nhim bnh hoi t gan ty, m trng v u vng. Trong 8 thng u nm 2012, mc thit hi nui tm trn a bn tnh Sc Trng n nay chim gn 50%, trong din tch thit hi do bnh m trng chim trn 40%. 2. Vi-rt m trng (white spot disease virus) 2.1. Hnh thi vi-rt Virion c chiu di dao ng t 210 n 380 nm, b dy t 70 n 167 nm. Phn ph ging nh ui nm mt u ca virion. Phn v ca dy 6-7 nm c cu to t mng lipit gm 2 lp trong sut c ngn cch bi 1 lp c. Phn nhn nm bn trong c cu trc mch vng. Kch thc ca nhn c khc bit gia cc isolate, chiu di t 180 n 420 nm v b dy t 54-85nm.
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Hnh 1. A) Hnh thi ca virion WSSV B) nh chp di knh hin vi in t cho thy phn ph ging nh ui (thc o 250 nm) (Duran v ctv, 1996) 2.2. B gen Genome ca WSSV l si i DNA dng vng, vi lng A + T chim 59%. Kch thc genome khc nhau gia cc isolate, Thi Lan 293 kbp, Trung Quc 305 kbp, i Loan kpb. Kt qu phn tch trnh t cho thy genome ca WSSV c cha t 531 n 684 khung c m (open reading frame, ORF) vi codon bt u l ATG. Trong , 181 - 184 ORFs m ha protein chc nng c kch thc t 51 n 6077 amino acid. Khong 21 - 29 % ORFs m ha protein WSSV. Cc protein ny gm nhng enzyme tham gia vo qu trnh tng hp nucleic acid v nhn bn DNA nh DNA polymerase, non-specific nuclease, tiu phn ca ribonucleotide reductase, thymidine kinase, thymidylate kinase. Cc protein c chc nng gi nh gm collagen - like protein, flagellin, chitinase

Hnh 2. Cu trc rt m trng (dng Thi Lan)

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tng qut b gen vi-

2.3. c im bnh l Tm b bnh c mu hng n hng , xut hin nhng m mu trng c ng knh t 0,53 mm mt trong lp v kitin vng u ngc v t bng th 5, 6 sau lan ra ton thn. ao nui, tm b bnh dt vo b v c du hiu bnh 1 n 2 ngy trc khi cht. T l cht c th ln n 100% trong vng 10 ngy k t lc bt u bnh

Hnh 3. Cc m trng xut hin trn v u ngc ca tm bnh 2.4. Cc con ng ly truyn Bnh m trng do WSSV ly lan rt nhanh qua 2 con ng chnh. Ly lan theo chiu dc, t b m nhim ly cho con. Ly lan theo chiu ngang, tm nhim vi-rt t ngun nc nui, t cua, cng mang vi-rt t ao ny sang ao kia, t dng c sn xut mang mm bnh, t tm cht, chim, c v tnh a vo ao. 2.5. Vt ch mang mm bnh WSSV c pht hin cm nhim nhiu loi vt ch khc nhau Penaeus monodon, P. japonicus, P. indicus, P. chinensis, P. merguensis, P. aztecus, P. stylirostris, P. vannami, P. duorarum v P. setiferus; tm cng xanh - Macrobrachium rosenbergii, cua Calappa lophos, Portunus sanguinolentus, Charybdis granulata v C. feriata. 2.6. Phng bnh Cho n nay, cng tc phng bnh vn l bin php hiu qu nht hn ch thit hi do bnh m trng. S phng nga gm c cc bin php sau: chn tm b m, tm ging c cht lng tt, khng nhim WSSV, ngun nc cp cho ao nui tm phi lng lc v kh trng, vt tm cht ra khi ao, ngn chn khng cho tm v gip xc khc vo ao nui, duy tr cc yu t mi trng thch hp cng nh cht lng thc n tt, khu phn n hp
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l. Nc nui tm b bnh WSSV phi x l bng Clorua vi nng (30 - 50 g/m3), khng c x ra ngoi. Khi pht hin bnh tt nht l thu hoch ngay. 2.7. Phng php chn on pht hin mm bnh WSSV c trn tm bng nhiu phng php khc nhau nh: phng php m hc xc nh bnh da trn cc th vi hin din trong t bo biu m; phng php lai ti ch (In situ hybridization) du hiu xc nh bnh l kt qu lai gia on gen dng lm mu d v on gen ca tc nhn gy bnh trong iu kin nghim ngt; phng php PCR xc nh bnh da trn l sn phm khuch i ca mt on gen c trng ca tc nhn gy bnh; phng php Dot blot v Southern blot c thc hin vi sn phm ca PCR; phng php min dch hunh quang du hiu nh bnh l kt qu kt hp c hiu gia khng nguyn v khng th, s dng knh hin vi in t.

Bi 2.LY TRCH DNA

1. Tng quan v phng php ly trch Bc 1: Ph v mng t bo v mng nhn. Thng thng ngi ta nghin t bo hoc m trong mt hn hp cht ty (SDS, sarcosyl, CTAB) v protein K. Hn hp ny s ph v mng t bo v mng nhn, gii phng DNA ra mi trng ng thi phn hy cc protein lin kt vi DNA. Bc 2: Loi b cc thnh phn khng mong mun, thng s dng dung dch phenol:chloroform Bc 3: Kt ta nucleic acid. Mc ch ca vic kt ta l nhm thu nhn nucleic acid di dng c c, mt mt nhm bo v chng khi s phn hy ca cc enzyme, mt khc c th ha tan chng li trong dung mi theo nng mong mun. Hai cch kt ta thng dng l dng ethanol hoc isopropanol. Trong c hai phng php, nucleic acid s c thu nhn li bng ly tm. Sau , cn kt ta phi c ra trong ethanol 70% loi b cc mui hoc cc du vt ca isopropanol cn dnh li. 2. Thc hnh 2.1. Phn nhm Sinh vin trong lp (20 - 30 sinh vin) c chia thnh 5 nhm (4 - 6 sinh vin/nhm). Mi sinh vin ly trch 1 mu. 2.2. Chun b mu, ha cht, dng c, thit b Mu: mu tm s, tm chn trng c mua tri ging hoc mua ti ch (sinh vin t chun b mu cho thc hnh) Ha cht: SDS (sodium dodecyl sulphate) v NaOH Dng c:
Kp, ko, n cn, khay ng nc , bt ghi mu.

Micropipet 100 - 1000 l. Thit b: My ly tm, bp in. Vt t tiu hao:

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 Chy v trng dng nghin mu trong tube ly tm

Tube ly tm 1,5 ml hp kh trng

u cn xanh, vng hp kh trng

Gng tay, khu trang, khn giy

2.3. Pha dung dch ly trch DNA th Dung dch ly trch DNA th c nng NaOH 0,025 N, SDS 0,0125% (theo Kiatpathomchai v ctv, 2001) c pha theo cc bc sau:
Pha 100 ml NaOH 1N (MW=40)

Cn 4 g NaOH ha trong 100 ml nc ct 2 ln Hp kh trng 121oC trong 20 pht

Pha 100 ml SDS 0,5% (MW=288,38)

Cn 0,5 g NaOH ha trong 100 ml nc ct 2 ln Hp kh trng 121oC trong 20 pht

200 ml 5 ml 5 ml 190 ml Nng cui NaOH 0,0025 N SDS 0,0125%

Dung dch ly trch th, thnh phn nh bng sau

Dung dch ly trch DNA th NaOH 1N SDS 0,5% H2O

2.4. Ly trch DNA DNA c tch chit theo nghin cu ca Kiatpathomchai v ctv (2001). Quy trnh c tin hnh nh sau:

Dng kp ly mang tm cho vo eppendorf 1,5 ml v thm 500 l dung dch Dng chy v trng nghin cho mu ng nht. Thm 300 l dung dch ly trch th DNA, tip tc nghin. un si eppendorf 100oC trong 20 pht (thy dung dch trong mu si ln). Lm lnh mu nhanh: chuyn eppendorf vo nc lnh trong 5 pht.

m ly trch th DNA (0,025 N NaOH v 0,0125% SDS).

Ly tm 9.000 vng/pht trong 5 pht. Ht dch ni vo eppendorf mi.

Nn thc hin phn ng PCR ngay sau khi tch chit. Tuy nhin c th gi mu 20oC trong thi gian ngn (khong 1 - 2 ngy).

Bi 3. CHN ON WSSV BNG NESTED - PCR

1. Tng quan v PCR 1.1. Nguyn tc ca PCR Phn ng PCR c thc hin trn c s sinh tng hp DNA theo nhiu chu k ni tip nhau, mi chu k gm 3 bc nh sau: bin tnh (denature), bt cp (annealing), ko di (extension)

Hnh 4. Cc bc trong phn ng PCR 1.2. Cc hn ch ca phng php PCR S ngoi nhim l vn ln nht Ngun ngoi nhim ln nht thng l sn phm khuch i ca nhng ln thao tc trc. C th khc phc 1 phn ca vn bng cc cch sau:
Thit lp phn ng v phn tch sn phm khuch i c tin hnh nhng a

im cch xa nhau
Dng c thit lp phn ng khng s dng vo cc thao tc khc. S dng u

tp c lc khi ht ha cht.
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 Dng tia t ngoi loi b cc phn t cn li t ln khuch i trc Chia nh cc thnh phn phn ng
S dng dUTP thay th cho dTTP. Trc mi phn ng, ngi ta thm vo

dung dch phn ng uracylglycosylase, enzyme ny phn hy tt c cc DNA c mang dUTP nhim t ln trc. ng thi, enzyme s b phn hy bi nhit ngay t ln bin tnh u tin. S nhn bn khng c hiu Xy ra khi mi bt cp khng c hiu vi nhng trnh t khc vi trnh t ch. Mt s phng php khc phc
Phng php hot start vi c im ch yu l trnh s tip xc ca cc thnh

phn quan trng trong phn ng trc khi trnh t ch c bin tnh hon ton.
Phng php nested PCR s dng thm 1 cp mi c thit k bt cp vi sn

phm khuch i ca ln th nht. Sai st do Taq polymerase gy ra S sao chp bi Taq polymerase cho t l sai kh cao (10-4, ngha l 10000 nucleotide th enzyme gn sai 1 nucleotide). c tnh ny khng nghim trng nu ta ch cn xem xt kch thc hay s c mt ca 1 sn phm khuch i, nhng c ngha ln nu cn xc nh chnh xc trnh t nucleotide. Khng th loi b hon ton cc sai st ny m ch c th gim bt bng cch m bo cn bng nng cc nucleotide trong phn ng, s dng cc DNA polymerase chu nhit c trung thc cao nh VentTM, Pfu, Ultma DNA polymerase. 2. Thc hnh chn on WSSV 2.1. Gii thiu on mi Nm 1996, Lo v ctv thit k 2 cp mi: 146F1/146R1 v 146F2/146R2, pht hin s hin din ca WSSV bng k thut nested - PCR. Cp mi 146F1/146R1 khuch i on bn ngoi vi kch thc 1447 bp. Cp mi 146F2/146R2 khuch i on bn trong vi kch thc 941 bp. Quy trnh ny c t chc th y th gii (OIE) cng nhn l quy trnh chun v khuyn co cc phng th nghim s dng chn on WSSV.

Tn primer 146F1 146R1 146F2 146R2

Trnh t 5-ACT-ACT-AAC-TTC-AGC-CTA-TCTAG-3 5-TAA-TGC-GGG-TGT-AAT-GTT-CTT-ACG-A-3 5-GTA-ACT-GCC-CCT-TCC-ATC-TCC-A-3 5-TAC-GGC-AGC-TGC-TGC-ACC-TTG-T-3

V tr ca primer trn trnh t trn GenBank AF440570 259206259229 260629260653 259457259479 260376260398

Kch thc 1447 bp 941 bp

Hnh 5. V tr ca primer 146F1/146R1, 146F2/146R2 v kch thc sn phm (Lo v ctv, 1996) 2.2. Phn nhm Sinh vin trong lp (20 - 30 sinh vin) c chia thnh 5 nhm (4 - 6 sinh vin/nhm). Mi mi nhm thc hin 1 mu. 2.3. Chun b ha cht, dng c thit b Ha cht thc hin PCR: Nc ct
PCR buffer 10 X Taq polymerase 5U/l MgCl2 25mM dNTP 10 mM Primer146F1/146R1,

146F2/146R2 10M DNA ly trch

Dng c, thit b
Micropipet 0,5 - 10 l, 10 - 100 l, 100 - 1000 l

u tp trng, vng, xanh Tube PCR 0,2 ml

My lun nhit, t cy v trng 10

 My nh, gng tay, khu trang, khn giy 2.4. Thc hin PCR bc 1 vi cp mi 146R1/146F1 2.4.1. Tnh th tch cc thnh phn phn ng cn ht Thnh phn phn ng cho 1 phn ng vi tng th tch l 25 l Thnh phn Nng u Nng cui Th tch cn ht H2O 16,7 l PCR Buffer 10X 1X 2,5 l MgCl2 25 mM 1,5 mM 1,5 l dNTP 10 mM 0,2 mM 0,5 l Primer 146F1 10 M 0,5 M 1,25 l Primer 146R1 10 M 0,5 M 1,25 l Taq polymerase 5 U/ l 1,5 U 0,3 l DNA khun 1 l tnh th tch PCR buffer, MgCl2, dNTP, primer cn ht, chng ta s dng cng thc: C1 x V1 = C2 x V2 C2: nng cui V2: tng th tch ca 1 phn ng C1: nng u V1: th tch cn ht V d: Cch tnh th tch primer cn ht.
Nng u ca primer l 10 M (C1 = 10 M) Nng cui ca primer trong 1 phn ng l 0,4 M (C2 = 0,4 M) Tng th tch ca 1 phn ng l 25 l (V2 = 25 l) S dng cng thc trn, suy ra th tch primer 10 M cn ht: V1 = 1l

tnh th tch Taq polymerase cn ht, chng ta s dng tam thc: 1 l Taq polymerase ? l Taq polymerase 5U Cn 1,5 U cho 1 phn ng

Sau khi tnh th tch cn ht cho 1 phn ng, chng ta tnh th tch cn ht cho n phn ng: ly th tch cho 1 phn ng * n. Lu : khng tnh i vi th tch DNA ly trch v phi tnh d thm 1 hoc 2 phn ng tr hao ht trong thao tc.
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Thnh phn

Nng u

Nng cui 1X 1,5 mM 0,2 mM 0,5 M 0,5 M 1,5 U

Th tch cn ht (l) 16,7 2,5 1,5 0,5 1,25 1,25 0,3 1

H2O PCR Buffer 10X MgCl 2 25 mM dNTP 10 mM Primer 146F1 10 M Primer 146R1 10 M Taq polymerase 5 U/l DNA khun 2.4.2. Thc hin mix mu

Th tch cn ht cho 6 phn ng (n=6) (l) 100,2 15 9 3 7,5 7,5 1,8 -

S dng micropipet pha master mix chung, bao gm cc thnh phn phn ng trong 1 tube ln (khng ht DNA khun). Sau chia u master mix vo n tube PCR 0,2 ml. Cui cng, ht 1 l DNA khun cho vo tube PCR 0,2 ml c tng th tch 25 l. Lu khi thc hin: - Mix mu trong t v trng, phi v sinh t trc khi s dng. - Khi mix mu, t cc tube ha cht trn gel hoc bo gi lnh cc ha cht - Cc ha cht nh H20, MgCl2, PCR buffer, dNTP, primer ng khi bo qun - 20oC nn phi r ng v trn u trc khi ht. - Taq polymerase rt d hng nn khi ly ra khi t m nn phi cho ngay vo master mix v ct li vo t m. - trnh tp nhim vo ha cht, nn ct ht cc ha cht trc khi m np cc tube cha DNA khun.

2.4.3. Thc hin chu trnh nhit Chu trnh nhit c ci t nh bng

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Nhit 940C 940C 550C 720C 720C 40C

Thi gian 4 pht 1 pht 1 pht 2 pht 5 pht

S chu k 1 40 1

2.5. Thc hin PCR bc 2 (nested PCR) vi cp mi 146F2/146F2 Sn phm PCR ca bc 1 tr thnh khun DNA cho PCR bc 2 khi thc hin vi cp mi 146F2/146F2. Cch tnh th tch thnh phn phn ng, thc hin mix mu, chu k nhit ging vi bc 1. Sau khi thc hin xong, sn phm PCR c in di kim tra ngay hoc tr t m. Lu : v bc 2 s dng sn phm PCR ca bc 1 lm khun nn kh nng b tp nhim t mu ny sang mu, t sn phm PCR vo ha cht l rt cao. Khi thc hin phi cn thn, phi ct ht ha cht, ng np ht cc tube cha master mix trc khi m np tube PCR 0,2 ml.

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Bi 4. IN DI TRN GEL AGAROSE

1. Tng quan v phng php in di trn gel agarose Agarose l mt trong cc dng ca polysaccharide. Cc nh sinh hc phn t khai thc c tnh th gel trong in di phn tch DNA. Agarose s to thnh cc ht agarose sau khi tan nhit cao hoc un si trong vi pht. Khi ngui li, nhng ht agarose s kt t li vi nhau. Gia cc ht c nhng l rt nh. Kch thc ca cc l ny x dch cht t theo nng ca agarose gel. Trong in trng, DNA di chuyn t in cc m sang in cc dng, v DNA mang in tch m. Khi DNA di chuyn qua cc l ca agarose, s c xt gia cc ht agarose v phn t DNA tao ra lc khng lm ngn cn s dch chuyn ca DNA. DNA c phn t cng ln th lc cn cng mnh, di chuyn cng chm v ngc li. Nh vy m chng ta c th phn tch c cc on DNA trn gel agarose. Sau khi in di trn gel, cc on DNA c phn ra ty theo trng lng phn t. Chng ta c th quan st chng bng mt nh k thut nhum mu DNA vi ethidiumbromide v chiu tia UV. 2. Thc hnh phn tch sn phm PCR trn gel agarose trong quy trnh chn on WSSV bng nested PCR 2.1. Chun b ha cht, dng c Agarose
Dung dch m TBE 0,5X B nhum ethidiumbromide Micropipet 0,5 - 10 l u cn trng Giy bc, gng tay, khu trang My nh

Loading dye
Khun gel v lc B in di

L vi sng 2.2. Tin hnh gel Sn phm PCR c phn tch trn gel agarose 1%. gel agarose 1%, tin hnh cch bc sau:
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Kim tra li s mu chn s ging trn gel t chn th tch khun Tnh lng agarose cn dng 100 ml dung dch TBE 0,5X 60 ml TBE 0,5X (th tch ca khun) S dng quy tc tam sut, lng agarose cn dng l 0,6 g. cn 1 g agarose ? g agarose

ph hp

gel agarose 1%

Cn ng lng agarose cn dng (0,6 g) cho vo chai c cha TBE 0,5 X (60 ml)

y np chai, lc u, t vo l vi sng, un 2 pht cho agarose tan ht.

vo khun, t lc vo to cc ging trn gel. i 40 pht cho gel ng, sau

g gel ra khi khun mt cch nh nhng, trnh lm v gel. C th tr gel trong TBE 0,5 X 2.3. Tin hnh in di
Trn u 5 l DNA ly trch hoc sn phm PCR vi 1 l loading dye. Sau ht

hn hp ny cho vo ging. in di di hiu in th 80 V, trong 30 pht. Nhum gel trong b ethidium bromide trong 30 pht. Sau quan st di tia UV. Ghi nhn kt qu bng my nh. 2.4. Phn tch kt qu kt lun mu xt nghim c DNA ca WSSV hay khng, chng ta da vo kt qu PCR bc 2 khi thc hin vi cp mi 146F2/146R2. Kt lun mu xt nghim c DNA ca WSSV khi: i chng dng c bng DNA (941 bp), i chng m khng c bng, mu xt nghim c bng c cng kch thc vi bng ca i chng dng (941 bp) Kt lun mu xt nghim khng c DNA ca WSSV khi: i chng dng c bng DNA (941 bp), i chng m khng c bng, mu xt nghim khng c bng c cng kch thc vi bng ca i chng dng (941 bp)

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V d:

Hnh 6. Kt qu in di sn phm khuch i khi thc hin nested PCR vi mu tm nghi ng nhim WSSV thu thp ti huyn m Di. Ging 1 v 9: i chng dng; ging 7 v 15: i chng m; ging 2, 3, 4, 5, 6 sn phm mu khuch i vi cp mi 146F1/146R1; ging 10, 11, 12, 13, 14, sn phm mu khuch i vi cp mi 146F2/146 R2; ging 8: thang DNA 1000 bp. in di vi gel agarose 1,5% di hiu in th 80 V trong 30 pht. (Thin Th Kim K, 2012) Kt qu PCR bc 2 khi thc hin vi cp mi 146F2/146R2 cho thy:

i chng dng c bng 941 bp i chng m khng c bng

5 mu xt nghim c bng cng kch thc vi i chng dng. Nn kt lun mu c DNA ca WSSV.

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TI LIU THAM KHO

1. Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng, S.E., Chen, Y.T., Chou, C.M., Yeh,

P.Y., Huang, C.J., Chou, H.Y.,Wang, C.H., Kou,G.H. 1996. Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis. Aquat. Org. 25, 133141.
2. Durand S., Lightner D.V., Nunan L.M., Redman R.M., Mari J & Bonami J.R.

(1996) Application of gene probes as diagnostic tools for white spot baculovirus (WSBV) of penaeid shrimp. Diseases of Aquatic Organisms. 27, 5966.
3. Kiatpathomchai1

Wansika,

Vichai

Boonsaeng,

Anchalee

Tassanakajon3,

Chainarong Wongteerasupaya, Sarawut Jitrapakdee, Sakol Panyim. 2001. A nonstop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon. Diseases of Aquatic Organisms. 47, 235239
4. Thin Th Kim K. 2012. Nghin cu pht hin vi-rt m trng (wssv) trn tm ti

tnh C Mau bng Nested - PCR. Kha lun tt nghip chuyn ngnh cng ngh sinh hc.

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