M T K THUT PCR (Polymerase Chain Reaction) TRONG CHUN

ON BNH VIRUS M TRNG TRN TM TH CHN TRNG

(Penaeus vannamei, Boone 1931) GIAI ON GING
TECHNICAL DESCRIPTION PCR (Polymerase Chain Reaction) IN DIAGNOSTIC VIRUS
WHITE SPOTS ON THE WHITE SHRIMP (Penaeus vannamei, Boone 1931) IN THE EARLY
STAGES VARIETY
Nguyn Vn Cng*, Pattarawan Chanakul
Phng Sau i hc Trng i hc Vinh
Email: htc48ts.dhv@gmail.com
ABSTRACT
In this paper, an effective PCR technique was described to diagnose the white spot syndrome
virus in white Shrimp fingerlings (Penaeus vannamei),( Boone 1931). The results also
provide some new findings to supplement some previously published results to improve the
accuracy of PCR technique to detect WSSV in Shrimp.
Keywords: PCR (Polymerase Chain Reaction), Penaeus vannamei, specimens.
T VN
Tm Th Chn Trng l loi tm hin ang
c nui nhiu trn din tch rng v
mang li ngoi t tng i ln cho ngnh
Thy sn Vit nam. Tuy nhin, loi tm ny
d cm nhim vi bnh virus m trng vi
cng cao. Vi rt gy bnh m trng
c xem l mt trong nhng tc nhn gy
cht tm hng lot nhiu m hnh nui
tm trn khp th gii. S cm nhim ca
loi vi rt ny trn nhiu loi vt ch/vt
mang mm bnh khc nhau (tm bin, tm
nc ngt, cua, tm tp hoang d) c
chng minh v cng b. WSSV tn cng
nhiu c quan khc nhau trn c th vt ch
v s hu c hai phng thc ly lan ngang Hnh 1. Loi Tm Th Chn Trng (Penaeus
vannamei, Boone 1931)
v dc.
Cho n hin nay, cc phng thc phng bnh hin ang c ng dng vn cha mang li
hiu qu cao. V vy, nghin cu c thc hin vi mc tiu m t thm v k thut PCR
trong chun on bnh virus m trng trn Tm Th Chn Trng giai on con ging sn
xut ti Cng ty C Phn Chn Nui C.P Vit Nam qua c th hiu r v phng thc
ly nhim ca loi vi rt ny v xut phng thc qun l bnh hiu qu hn. PCR c
s dng trong cc nghin cu sinh hc v y hc phc v nhiu mc ch khc nhau, nh pht
hin cc bnh di truyn, nhn dng, chun on nhng bnh nhim trng, tch dng gen v
xc nh huyt thng.
VT LIU V PHNG PHP NGHIN CU
i tng v vt liu nghin cu
i tng nghin cu

354

Loi Tm Th Chn Trng (Penaeus vannamei, Boone 1931)

cc giai on Nauplius v Postlarvae c ng ti Tri sn xut
ging thuc Cng ty C Phn Chn Nui C.P Vit nam Chi
nhnh Bnh nh 3.

Hnh 2. Tm Th Chn
Trng giai on thu mu
nghin cu
Vt liu nghin cu
Mu Nauplius v mu Postlarvae cha nhim bnh l.
a im v thi gian nghin cu
Cc nghin cu c tin hnh thc hin ti Phng PCR thuc Cng ty C Phn Chn Nui
C.P Vit nam Chi nhnh Bnh nh 3, thn Xun Thnh x M An huyn Ph M tnh Bnh
nh. Thi gian nghin cu tin hnh t ngy 20/1/2012 n ngy 20/10/2012.
Phng php nghin cu
Phng php thu mu
i vi mu Nauplius: Chun b dng c gm que mi nhn, Microcentrifuge 1.5ml c cha
800l cn 960, vt c 54T v gng tay y t. Phng php thu mu Nauplius l dng vt vt
Nauplius quanh thng su 10 15 cm sau dng que ly khong 500l Nauplius cho
vo ng nghim cha cn.
i vi mu Postlarvae: Chun b dng c gm vt c 12T, chu nha 2 2,5 lt, ti ng
tm, dy thun. Phng php thu mu Postlarvae l dng vt ly su 10 20 cm, 4
im trong b, cn thc hin khuy nc tch ly nhng con yu (600 800 con), sau
cho vo ti ng tm v thm 2 lt nc, tht ming ti v khng bm xy, cui cng ngm
thit b dng chung vo soludine 50% t nht mt pht v gi mu khong 24 gi.
Phng php nghin cu
Pht trin, b sung k thut PCR s dng trong chun on bnh WSSV trn Tm Th Chn
Trng da trn k thut PCR cng b.
Phng php x l s liu
Cc s liu thu thp c x l trn my chy PCR v c kt qu trn my tnh. Ton b s
liu c x l theo phng php thng k sinh hc trn phn mm Microsoft Excel v phn
mm SPSS 16.0.
KT QU NGHIN CU V THO LUN
Pht trin cc khi nim v k thut PCR
PCR l Polymerase chain reaction c ngha l phn ng tng hp DNA nhn to da vo tnh
c hiu ca cp mi chuyn bit cho on DNA ch v cc chu k nhit. PCR l mt k
thut ph bin trong sinh hc phn t nhm khuych i (to ra nhiu bn sao) mt on
DNA m khng cn s dng cc sinh vt sng.
M t quy trnh chun on bnh virus m trng bng k thut PCR trn ging Tm
Th Chn Trng (Penaeus vannamei, Boone 1931)
M hnh chun cho k thut PCR trn ging Tm Th Chn Trng (Penaeus vannamei,
Boone 1931):
355

Hnh 3. M hnh chung cho k thut chy PCR

Chun b mu chy PCR v cc bc lm PCR
Cc dng c cn thit gm mung nha, chu nha, Microcentrifuge 1.5 ml c 800l cn 960,
gi nha c nh dng cao, vi mn, nc ct, gng tay y t, kp. Lc tm bng vi mn, phun
ra bng nc ct. Dng mung nha ly tm vo microcentrifuge 1 ng cn. Ha cht IQ
2000 WSSV dng cho 1 mu:
Phn ng th nht (First): First PCR PreMix 7.5 l, IQzyme DNA Polymerase 0.5 l. Total
8.0 l.
Phn ng th 2 (Nested): Nest PCR PreMix 14 l, IQzyme DNA Polymerase 1 l, Total 15
l.
K thut pha mu: u tin ht 10l positive standards 104 vi 90l dung dch Yeast tRNA
ta c positive standards 10. Tip theo thc hin ht 10l positive standards 10 vi 90l
dung dch Yeast tRNA ta c positive standards 10 v cui cng ht 10l positive standards
10 vi 90l dung dch Yeast tRNA ta c positive standards 10.
Tin hnh pha ha cht cho phn ng First: Trc ht l ht 2l nc ct vo tube 0.2 ml
lm mu chng m. Ln lt ht 2l cht ha tan DNA (RNA) vo tube 0.2 ml chun b sn
v chun b ha cht nh sau: S mu chy = s lng mu + mu chng m + mu chng
dng + mu khc. Tip tc ht 8 l ha cht First vo tube 0.2 ml cha DNA (RNA), thc
hin vortex 30 giy v tr s 2000 ca my v ly tm nhanh 30 giy (khong 7500 vng quay
356

xy ra) v tin hnh cho vo my PCR v chn chng trnh. Cn ghi thng tin chy my vo
s theo di tin kim sot qu trnh chy mu nhm em li hiu qu chnh xc cao nht.
Tin hnh pha ha cht cho phn ng Nested: i vi phn ng ny cn chun b ha cht
trong t v khun ht 15l vo tube 0.2 ml mu chy phn ng Fisrt, Vortex 30 giy v
tr s 2000 ca my v ly tm nhanh 30 giy (khong 7500 vng quay xy ra). Sau cho vo
my PCR v chn chng trnh. Cui cng cn ghi thng tin chy my vo s theo di.
M t chng trnh chy my PCR: Thc hin First PCR gm cc thng s 940C 30sec,
62 0C 30sec, 720C 30sec, repeat 5 cycles, v 94 0C 15sec, 620C 15sec, 720C 20sec, repeat 15
cycles, cui cng l dy s 720C 30sec, 200C 30sec 40C
. Tip theo thc hin Nested PCR
gm dy s 94 0C 20sec, 620C 20sec, 72 0C 30sec, 25 cycles, v 720C 30sec, 20 0C 30sec 40C
at the end of the final cycle.
Qu trnh tch chit DNA trong k thut PCR
Trong bc ny k thut tch chit DNA c m t ln lt nh sau: Ly vi lng mu
(300l) cn thc hin cho vo ng ly tm 1.5 ml cha 600l Lysis buffer. Sau tin hnh
nghin mu tm trn khay 2 3 pht sau votex 2 3 pht v tr s 4 ca my, i vi
Nauplius votex 10 pht v tr s 8 ca my. Tip theo nu mu 95 1000C trong 10 pht
(lm lnh khong 5 pht). Tip n cn ly tm 12000 rpm trong 10 pht. Tip theo ht 200l
dch trong pha trn vo ng cha sn 400l Absolute ( lm lnh) v thc hin trn u hn
hp ny. Tin hnh ly tm 12000 rpm trong 5 pht. Sau Absolute v phi ng trong 30
pht v thm nc ct tit trng. Tin hnh hp 75 0C trong vng 10 pht v ly tm
12000 rpm trong 10 pht v cui cng ca qu trnh l ht 2 l DNA tinh sch cho vo tube
0.2ml chy phn ng PCR. Nguyn tc ca vic tch chit DNA l da trn s phi hp c
hc, ha hc v sc nhit tch chit DNA virus ra dch chit.
Qu trnh khuych i DNA trong k thut chy PCR
PCR (polymerase chain reaction) l phn ng tng hp DNA nhn to da vo tnh c hiu
ca cp mi chuyn bit cho on DNA ch v cc chu k nhit.
Thnh phn ca phn ng
gm DNA bn mu, mi xui
v mi ngc, dNTP (gm 4
loi: dATP, dGTP, dTTP,
dCTP), Enzyme polymerase
(Taq polymerase), dung dch
m, nc ct 2 ln ( kh
trng). Trong DNA hay
acid nucleic ch, tc l chui
acid nucleic. (V d, on
acid nucleic c hiu ca vi
khun gy bnh, ca gene
bnh l, ca du n di
Hnh 4. Hnh m phng kt qu trn my tnh v mi xui v truyn,) m phn ng PCR
mi ngc
s khuych i ln chng
c th c pht hin trong
bnh phm.

357

Phn ng PCR s khng xy ra c nu bnh

phm khng c nucleic acid ch. Mt trng hp
khc l trong bnh phm c nucleic acid ch,
nhng do bnh phm c sa son khng thch
hp hoc khng ng cch nn nucleic acid ch
khng bc l ra, hoc trong bnh phm hy cn
cc cht c ch phn ng PCR. Trong nhng
trng hp ny kt qu PCR s m tnh, nhng l
m tnh gi. V vy vn sa son bnh phm
cho th nghim PCR phi c coi trng, c
bit trong cc phng th nghim p dng PCR
Hnh 5. M phng on khi u v
chun on pht hin bnh l.
on kt thc ca chui DNA ch
Primer hay on mi, tc l nhng on DNA n (oligonucleotide) c kch thc ch vi
chc base (18 30), c th bt cp theo nguyn tc b sung vo on khi u v on kt
thc ca chui DNA ch khi chui ch ny c bin tnh thnh si an.
Trong th nghim PCR, on mi c hai vai tr
chnh : Quyt nh nn tnh c hiu ca th
nghim, v nu on mi c chn cng c
hiu cho chui ch, ngha l ch c th bt cp
trn chui ch m khng th bt cp c trn
cc chui DNA khc ngoi chui ch, th sn
phm PCR cng c hiu v th nghim PCR
cng c hiu. Khi ng men polymerase v men
polymerase ch c th bt u tng hp si b
sung cho chui DNA ch mt khi n nhn dng
c u 3, (l u m n xc tc cho mt dNTP
c gn vo) ang tnh trng si i. Thng
Hnh 6. M phng cc on mi th thng trong phn ng PCR, ngi ta dng mt
nghim PCR
cp mi, gi l primer set, trong c mt mi
ln gi l up stream primer v mt mi xung gi l down stream prime. Cp mi ny
quyt nh nn kch thc ca sn phm PCR. Chng cng bt cp trn chui ch xa nhau
bao nhiu th kch thc ca sn phm PCR cng ln by nhiu v ngc li, cng gn nhau
bao nhiu th kch thc cng nh by nhiu. ng lu l dNTP deoxy nucleoside
triphosphate, tc l n v c th tng hp c cc bn sao ca DNA ch.

Hnh 7. M phng free nucleptides

Hnh 8. M phng taq polymerase

DNTP c cu to gm mt ng deoxyribose c gn mt base, c th l adenine (dATP) hay

thymine (dTTP) hay Cytosine (dCTP) hay guanine (dGTP), carbone s 1 (C1). Ba phn t
358

phosphate (triphosphate) c gn ti carbone s 5 (C5) ca phn t deoxyribose ny v y

chnh l ni m dNTP gn vo u 3 ca chui b sung trn chui ch. Nng lng cho
phn ng ny xy ra c ly t cc ni phosphate giu nng lng ca triphosphate trn
dNTP. cng chnh l l do ti sao phi l dNTP ch khng phi l dNDP (diphosphate)
hay dNMP(monophosphate). Men polymerase, phi l men polymerase chu c nhit .
Ngy nay c nhiu loi polymerase chu c nhit c ly trch hoc tng hp, ty
mc ch s dng m chng ta c th chn polymerase thch hp. Thng dng nht trong
cc phng th nghim l men Taq polymerase. L men polymerase trch t vi khun Thermus
aquaticus, l cc vi khun sng c trong cc sui nc nng. Khuych i DNA l mt
chui nhiu chu k, mi chu k gm 3 bc:
Bc 1, Bin tnh (denaturation): DNA c bin tnh nhit cao, thng l 94 950C,
trong thi gian 30s 1 pht.

Hnh 9. M phng Step 1

PCR l mt th nghim nhm khuych i chui nucleic acid ch thnh hng t bn sao
sau c th pht hin c. thc hin c vic khuych i acid nucleic ch, th
nghim PCR da vo nhng chu k nhit m mi chu k c 3 bc (hnh 9), mi bc ko
di khong vi chc giy n vi pht, tun t nh sau : u tin l nhit c nng ln
khong 94 0C lm bin tnh nucleic acid ch t dng si i (dsDNA) thnh si n
(ssDNA), y l giai on lm bin tnh (denaturation) DNA ch.
Bc 2, Bt cp mi (hybridization): Nhit cn cho s bt cp mi vi DNA mch khun
l khong 50 600C.

Hnh 10. M phng Step 2

K l giai on bt cp (anealing), lc ny nhit trong bung PCR h xung khong
55 0C cc on mi (primer) bt cp theo nguyn tc b sung vo hai u ca chui nucleic
acid ch, y l giai on quyt nh nn tnh c hiu th nhng sn phm PCR (PCR
product) s c hiu.
Bc 3, Ko di (elongation): Nhit c tng ln n 72 0C, DNA polymerase hot
ng ko di mch.

Hnh 11. M phng Step 3

359

Cui cng l giai on ko di (extension), lc ny nhit c nng ln khong 72 0C l

nhit ti ho men polymerase chu nhit xc tc phn ng tng hp bn sao ca DNA
ch theo nguyn tc b sung trn khun l cc si n (ssDNA) ch c on mi bt
cp vo trc ( giai on bt cp). S tng hp ny cn phi c s hin din cc deoxy
nucleoside tri phosphate (dNTP).
Qu trnh in di v phn tch kt qu trong k thut PCR
Qu trnh chun b TBE 10X v TBE 1X l mt cng vic i hi thc hin cn thn, chi tit
bi v n lin quan ti kt qu cui cng m k thut PCR chy c v cho ra kt qu chnh
xc hay khng ph thuc khng nh vo giai on chun b ny. Trc ht i vi chun b
TBE 10X chng ta s dng hp phc ha cht vi cng thc tng qut gm Tris HCl 108g +
Acid Boric 55g + EDTA 7,44g, sau cho nc ct vo khuy tan ha cht cho va lng
1 lt. Ri em Auto tit trng 121 0C 15 pht ch xong bc ny. Cn i vi vic chun b
TBE 1X: u tin ong 100ml TBE 10X, tip theo ong 900ml nc ct v cui cng lc
trn u thu c 1 lt dung dch TBE1X. Cn thc hin cn trng v lc tht u dung dch
ha tan theo k thut ca PCR. Sau qu trnh chun b TBE 10X v TBE 1X l n qu
trnh chun b gel, cng ln lt ong 110ml TBE 1X cho vo bnh tam gic b sung thm 2g
agarose cho vo bnh tam gic, lc u v sau nu trong l vi sng 2 pht, ly ra lc u,
nu tip 1 pht, lc u ri ngui ti 65 70C. n y ta thm 5l SafeView vo bnh
tam gic lc u. Tip theo gel vo khay ch 1 2 pht cho lc vo v cui cng
ngui nhit phng khong 20 30 pht. Khi thc hin xong cc bc ny th chng ta
tin hnh in di nh sau: Trc ht cn pha dung dch in di gm 100ml TBE1X thm 5l
SafeView. Sau cho tm gel vo my in di c cha dung dch in di. Thc hin ht 5l
dung dch loading v sn phm PCR trn u, sau ht 5l dung dch vo cc ging trn
tm gel. Lu mu postlarvae c ht sau cng v tr hai u tm gel cng vi mu
marker. Cn thc hin chn iu kin in di bng dng in l 100A/1 my, 110V, trong
thi gian 30 45 pht. Cui cng ca k thut PCR l thc hin chp nh da trn kt qu
phn tch v in di sn phm. Ta dng nc ct lau my chp nh. Sau cho tm gel vo,
t s v tn bnh l. Tin hnh bt n UV v chp nh.
c kt qu ca k thut PCR da vo hnh nh sau:

Hnh 12. Bng m ha kt qu ca qu trnh chy PCR

Lane 1: Sample of severe WSSV infection
Lane 2: Sample of moderate WSSV infection
Lane 3: Sample of light WSSV infection
Lane 4: Sample of very light WSSV infection
Lane 5: WSSV negative sample
Lane 6: Negative control (yeast tRNA or ddH2O)
Lane 7: WSSV P(+) standard, 2000 copies/reaction
Lane 8: WSSV P(+) standard, 200 copies/reaction
Lane 9: WSSV P(+) standard, 20 copies/reaction
Lane M: Molecular weight marker, 848 bp, 630 bp, 333 bp

KT LUN V KIN NGH

Bc u m t li qu trnh s dng k thut PCR chung v tin hnh pht trin, b sung k
thut ny vo trong cng vic chun on bnh virus m trng trn Tm Th Chn Trng
360

giai on ging. a ra c cc bc thc hin v m t tng i hon chnh cc chi tit

trong k thut chy PCR. Gii quyt c tnh cp thit ca thi im hin ti cng nh
nghin cu b sung mi mt s vn khi s dng k thut PCR trong lnh vc thy sn. Cn
thc hin cc nghin cu tng th v k thut PCR trn cc i tng khc trong ngh nui
thy sn nhm gim c nhng thit hi ng tic v bnh l ca chng.
TI LIU THAM KHO
Nguyn n, Hong Dn, L Vit Ly, Nguyn Thin, Trn Xun Th, 1983. Di truyn hc
ng vt. NXB Nng nghip, H Ni.
Nguyn Kim ng, 1998. Bi ging Di truyn hc. Dng cho ngnh nng nghip, chn nui
th y, nui trng thy sn, Trng H Nng Lm Hu.
Nguyn Minh Hon, Nguyn Kim ng, Phm Khnh T, 2000. Di truyn hc ng vt.
NXB Nng nghip, H Ni.
L nh Lng, Phan C Nhn, 1994. C s di truyn hc, NXB Gio dc, H Ni.
Trn nh Min, Phan C Nhn, 1981. Di truyn hc i cng. NXB Nng nghip v NXB
Mir Mat c va.
Nguyn Hng Minh, 1999. Di truyn hc. NXB Nng nghip, H Ni.
Hutt F. B, 1978. Di truyn hc ng vt. NXB Khoa hc v K thut, H Ni.
Douglas Tave, 1986. Genetics for Fisshery Managers; AVI Publishing Company, Inc.
Westport Connecticut.
De Robertis E.D.P, Nowinski W.W., Saez F.A, 1965. Biologia Celular. Sexta edicion
totalmente renovada, Edicion Revolucionada, Instituto del Libro, Vedado Habana, Cuba.
Falconer D.S, 1981. Introduction to Quantitative Genetics. Second edition, Longman London
and New York.
Klug W.S., Cummingham M.R, 1986. Concepts of Genetics. Scott, Foresmen, and Company
Glenview, Illinois, London England.

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