B GIO DC V O TO

TRNG I HC NNG LM THNH PH H CH MINH

B MN CNG NGH SINH HC

KHO LUN TT NGHIP

PHT HIN VIRUS BNH DCH T HEO

DA TRN ON GEN NS5B

Ngnh hc: CNG NGH SINH HC

Nin kho: 2003 2007
Sinh vin thc hin: NG TH THU NGN

Thnh ph H Ch Minh
Thng 9/2007

B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
B MN CNG NGH SINH HC

KHO LUN TT NGHIP

PHT HIN VIRUS BNH DCH T HEO

DA TRN ON GEN NS5B

Gio vin hng dn:

Sinh vin thc hin:

PGS.TS. TRN TH DN

NG TH THU NGN

KS. VNG H V
KS. LNG QU PHNG

Thnh ph H Ch Minh
Thng 9/2007

LI CM T
Trc tin con xin gi ngn li cm n n ba m knh yu, ba m lun hy
sinh dnh nhng g tt p nht cho con. Con xin cm n tt c ngi thn
lun yu thng, quan tm v lun c v cho con hc tp.
Em xin chn thnh cm n:
Ban gim hiu trng i hc Nng Lm thnh ph H Ch Minh, Ban ch
nhim B Mn Cng ngh sinh hc, cng tt c qu thy c truyn t
nhng kin thc qu bu cho em trong sut 4 nm hc.
Ban gim c cng tp th cn b Trung tm Phn Tch Th Nghim trng
i hc Nng Lm thnh ph H Ch Minh to iu kin thun li v tn
tnh gip em trong sut thi gian thc tp
Em xin trn trng bit n c Trn Th Dn v thy Nguyn Ngc Tun ht
lng hng dn v to iu kin thun li nht cho em trong sut qu trnh thc
hin ti tt nghip.
Em xin chn thnh cm n anh Vng H V, anh Lng Qu Phng
lun ch dn tn tnh cho em trong sut qu trnh thc tp.
Em xin gi li cm n n:
Thy Pht, anh t, ch Hng, ch Trang, cng vi cc anh ch Trung tm
lun gip , ng vin em trong nhng lc kh khn.
Cc bn lp CNSH K29 thn yu lun cng ti chia x, c v v gip
nhau trong sut thi gian thc tp tt nghip.

iii

TM TT KHA LUN
Ng Th Thu Ngn, i hc Nng Lm TP. H ch Minh, thng 9/2007,
PHT HIN VIRUS DTH DA TRN ON GEN NS5B.
Hng dn khoa hc:
PGS. TS. Trn Th Dn
KS. Vng H V
KS. Lng Qu Phng
ti c tin hnh t ngy 01 thng 03 nm 2007 n ngy 01 thng 08
nm 2007 ti Trung tm Phn tch Th nghim Ho sinh trng i hc Nng Lm
thnh ph H Ch Minh.
Gen NS5B l mt trong nhng gen ca b gen virus DTH ngy cng c
s dng nhiu trong nhng nghin cu xc nh chng, phn loi di truyn virus
DTH. Do , vic pht hin virus DTH bng phng php RT-PCR khuch i
on gen NS5B nhm ng dng vo cng tc chun on bnh v c th c s
dng hnh thnh d liu di truyn nh du dch t virus.
Kt qu t c nh sau:
1. Xc nh c quy trnh tch chit RNA thch hp t mu lch. Nng
LiCl 2,5 M cho ta RNA vi tinh sch v cht lng sn phm khuch
i cao.
2. a ra c quy trnh RT-PCR mt bc pht hin gen NS5B ca virus
DTH vi nng Taq gim t 2,5 UI xung 2 UI nhng cht lng sn
phm khuch i khng thay i.
3. Mc t s OD ca ELISA ln hn 2 cho kt qu RT-PCR dng tnh cao.
4. Kt hp c nhng c im bnh tch thng gp heo nhim bnh DTH
vi nhau thng qua kt qu chn on RT-PCR dng tnh.

iv

SUMMARY
Ng Th Thu Ngn, Department of Biotechnology, Nong Lam University,
HCMC. September, 2007. The title of thesis: DETECTION OF CLASSICAL
SWINE FEVER VIRUS BASED ON NS5B GENE.
Thesis were carried out from 01/03/07 to 01/08/07 at Biochemical Analysis
and Experimental Center, Nong Lam University.
One of the genes of CSFV genome is NS5B gene, which is widely used for
research on CSFV isolation and genetic typing. Therefore, CSFV detecting based
on amplification of the NS5B gene by RT-PCR method for the purpose of disease
diagnostic application and this process may be used for the generation of genetic
sequence data to be used for epidemiological tracing of virus.
Results obtained from the study:
(1) Determined RNA extracted protocol suitable for spleen sample. At
LiCl 2,5 M concentration, we collected higher pure RNA precipitate
which was used in RT-PCR resulted in highly qualitative product.
(2) Advanced single RT-PCR protocol to detect CSFV NS5B gene with
Taq concentration decreased from 2,5 UI down 2 UI, but RT-PCR
product quality was not different.
(3) OD ratio of ELISA was more than 2 value resulted in high positive RTPCR diagnostic.
(4) Combined symptoms of CSFV infected pigs together through positive
RT-PCR diagnostic result.

MC LC
NI DUNG

TRANG

Ba .............................................................................................................................. i
Trang ta ................................................................................................................... ii
Li cm t................................................................................................................. iii
Tm tt kha lun..................................................................................................... iv
Summary ....................................................................................................................v
Mc lc..................................................................................................................... vi
Danh sch cc ch vit tt ...................................................................................... viii
Danh sch cc bng .................................................................................................. ix
Danh sch cc hnh v biu ...................................................................................x
Chng 1. M U ................................................................................................1
1.1. t vn ..........................................................................................................1
1.2. Mc tiu v yu cu ...........................................................................................2
1.2.1. Mc tiu ...........................................................................................................2
1.2.2. Yu cu.............................................................................................................2
Chng 2. TNG QUAN TI LIU .....................................................................3
2.1. Bnh DTH ..........................................................................................................3
2.2. Mt s bnh tch c trng ca bnh DTH ........................................................5
2.2.1. DTH in hnh..................................................................................................5
2.2.2. DTH khng in hnh ......................................................................................6
2.3. Cu trc b gen virus DTH ................................................................................6
2.4. Mt s phng php chn on trong phng th nghim ..................................9
2.5. S lc cc nghin cu lin quan ....................................................................15
Chng 3. NI DUNG V PHNG PHP TIN HNH .............................17
3.1. Thi gian v a im ......................................................................................17
3.1.1. Thi gian ........................................................................................................17

vi

3.1.2. a im .........................................................................................................17
3.2. Ni dung nghin cu ........................................................................................17
3.3. Vt liu v ha cht ..........................................................................................17
3.3.1. Vt liu nghin cu ........................................................................................17
3.3.2. Ha cht .........................................................................................................17
3.4. B tr th nghim ..............................................................................................19
3.5. Phng php tin hnh .....................................................................................21
3.5.1. Thu thp mu bnh phm ...............................................................................21
3.5.2. Ly trch RNA tng s .....................................................................................21
3.5.3. Phn ng RT-PCR..........................................................................................24
Chng 4. KT QU V THO LUN ............................................................27
4.1. Kt ta RNA cc nng LiCl khc nhau ...................................................27
4.2. Thay i nng Taq trong phn ng. ............................................................28
4.3. So snh t s OD ca ELISA vi kt qu RT-PCR ..........................................30
4.4. Mi lin h gia kt qu RT-PCR vi c im bnh tch ..............................32
Chng 5. KT LUN V NGH .................................................................34
5.1. Kt lun ............................................................................................................34
5.2. ngh .............................................................................................................34
TI LIU THAM KHO .....................................................................................35
Ti liu ting Vit.....................................................................................................35
Ti liu ting Anh.....................................................................................................36
PH LC ................................................................................................................38

vii

DANH SCH CH VIT TT

BDV

: Border disease virus

BVDV

: Bovine viral diarrhoea virus

cDNA

: Complementary deoxyribonucleotide acid

ctv

: cng tc vin

DEPC

: Diethyl pyrocarbonate

DTH

: Dch t heo

EDTA

: Ethylendiaminetetraacid acetic

ELISA

: Enzyme linked immunosorbent assay

dNTP

: Deoxyribonucleoside triphosphate

IRF-3

: IFN regulatory factor 3

IFN

: Interferon

M-MLV

: Moloney murine leukemia virus

MOPS

: [N-morpholino] propanesulfonic acid

3NTR

: 3 nontranslated

NSP

: Nonstructural protein

PBS

: Phosphate buffered saline

OD

: Optical density

OIE

: Office International des Epizooties

RNA

: Ribonucleic acid

RT-PCR

: Reverse transcriptase polymerase chain reaction

SP

: Structural protein

Taq

: Thermus aquaticus

UV

: Ultra violet

UI

: Unit

viii

DANH SCH CC BNG

Bng 3. 1: Thnh phn phn ng RT-PCR ..............................................................25
Bng 4. 1: T s OD v hm lng RNA cc nng LiCl .................................27
Bng 4. 2: Kt qu RT-PCR 2 nng Taq. ........................................................29
Bng 4. 3: T s OD ca ELISA v kt qu RT-PCR ..............................................30
Bng 4. 4: So snh cc mc OD ca ELISA v RT-PCR ........................................31
Bng 4. 5: c im bnh tch cc mu RT-PCR dng tnh v m tnh ...............32

ix

DANH SCH CC HNH V BIU

Hnh 2. 1: Cu trc b gen Pestivirus ........................................................................7
Hnh 2. 2: Phn ng RT-PCR ..................................................................................12
Hnh 4. 1: Sn phm RT-PCR ca mu kt ta cc nng LiCl........................28
Hnh 4. 2: Sn phm RT-PCR 2 nng Taq ......................................................29
Hnh 4. 3: Kt qu sn phm RT-PCR cc mu bnh phm . ..................................33
Biu 4. 1: T s OD v hm lng RNA cc nng LiCl. ............................27

Chng 1
M U
1.1. T VN
Dch t heo (DTH) l mt trong nhng bnh gy thit hi kinh t ln nht
cho ngnh chn nui heo nc ta ni ring v cc nc trn th gii ni chung.
Nm 1997, bnh quay tr li ngay trn cc nc thnh vin ca Lin hip chu
u m h ngh bnh c thanh ton trit trc (Nguyn Tin Dng,
1999)
DTH l bnh rt d ly, s ly lan ch yu l do s tip xc gia nhng heo
sng vi nhau hoc vi nhng sn phm ca heo bnh, hoc nhng nguyn nhn
khcTc nhn gy bnh l mt virus thuc chi Pestivirus, h Flaviviridae. Virus
ny c tnh khng nguyn chung vi virus gy bnh tiu chy b (BVDV- Bovine
viral diarrhoea virus) v virus bnh bin gii cu (BDV- border disease virus).
Trong cng tc chn on hay phng v tr bnh, cc phng php truyn
thng da trn dch t lm sng hin ngy cng bc l nhiu khim khuyt khng
th khc phc v khng th theo kp tnh hnh hin nay nht l mm bnh cng ngy
cng kh kim sot. Mt khc, sinh hc phn t tuy mi t nn mng khong vi
thp k nhng c nhng bc pht trin vt bc vi nhiu thnh tu to ln, ,
ang v s ng gp vo mi mt ca i sng. Trong th y, sinh hc phn t vi
trng tm l cng ngh di truyn c ng dng kh sm trong chn on,
phng v tr bnh trn nhiu i tng gy bnh, trong c dch t - mt trong 4
bnh ca ngnh chn nui Vit Nam.
phn bit chnh xc virus gy bnh DTH vi nhng virus cng chi
Pestivirus nh BVDV, BDV, k thut RT-PCR tr nn hu ch da trn cc on
gen nh E2 (Wirz, 1993; Harding, 1994; Rugg Li, 1996; Knepp, 2003; Risatt, 2002,
2004; Pereda, 2005). Paton v cng s (2000) phn tch ba on gen (E2, 5NTR,

2
NS5B) trong vic phn bit chng virus DTH v tng kt s phn bit chng virus
DTH nhiu ni trn th gii k c chu ; kt qu cho thy mi quan h gia cc
chng mt s nc.
Trong nhng nm gn y, Vit Nam cng c rt nhiu nghin cu v
virus DTH ca cc tc gi nh Bi Ngha Vng v ctv (2003), Nguyn Th
Phng Duyn v ctv (2001), Kim Vn Phc v ctv (2004), Nguyn Th Vinh v
ctv (2004), H Thu Hng v ctv (2004) tuy nhin cha c nghin cu xc nh
trnh t nucleotide ca nhiu on gen khc nh NS5B, 5NTR ca nhng virus
phn lp c t cc dch. Gen NS5B l mt trong nhng gen ca b gen virus
DTH c s dng nhiu trong nhng nghin cu xc nh chng, phn loi di
truyn virus. V vy vic thc hin ti Pht hin virus DTH da trn on gen
NS5B nhm ng dng vo cng tc chn on bnh v to tin cho nhng
nghin cu xc nh chng, phn loi di truyn virus DTH sau ny.

1.2. MC TIU V YU CU
1.2.1. Mc tiu
Xc nh quy trnh RT-PCR pht hin virus DTH da trn on gen NS5B.
1.2.2. Yu cu
Xc nh triu chng v bnh tch ca heo bnh, thu nhn mu bnh phm.
Ly trch RNA tng s.
Chy RT-PCR theo dn liu ca Paton v cng s (2000) nhn vng gen NS5B.
So snh kt qu RT-PCR vi kt qu ELISA ca mu bnh phm.

Chng 2
TNG QUAN TI LIU
2.1. BNH DTH
2.1.1. Gii thiu
DTH l mt bnh quan trng nm trong danh sch loi A ca OIE. Nhng
bnh thuc trong danh sch A c nh ngha nh l nhng bnh truyn nhim c
kh nng ly lan rt nguy him v nhanh chng, bt chp bin gii quc gia, tr
thnh hu qu nghim trng i vi kinh t x hi v sc kho cng ng, tr nn
quan trng chnh trong vic kinh doanh th v nhng sn phm t chng (OIE,
2001).
Tnh hnh nhim bnh DTH trn th gii
DTH c m t u tin vo nm 1833 Ohio Hoa K. Nhng nm sau
, mt bnh trn heo Chu u bit nh l chng st trn heo ging y ht nh
bnh m t Hoa K. Cui th k 19, DTH phn tn khp ni trn th gii.
Vi s gia tng hiu bit v nguyn nhn v dch t hc bnh DTH, nhiu
quc gia thnh cng trong vic tit tr virus bng cch a ra nhng bin php
tng i n gin nh lut vn chuyn th v s ngn cm cho th n thc n
cha x l nh an Mch (1933), Phn Lan (1956), c (1963), Canada (1964),
Thy S (1974) v M (1977).
Nm 1997 in du m nt trong tm tr cc nh dch t hc, virus hc,
nhng cn b th y v nhng ngi hot ng trong ngnh chn nui heo: bnh
DTH, mt bnh m Lin hip chu u tng ngh l c thanh ton quay tr
li ngay trn cc nc thnh vin ca Lin hip (tnh n ngy 31/12/1997 c 424
dch ti H Lan). Nm nc khi EU (c, H Lan, Italia, Ty Ban Nha v B) tr
thnh nn nhn ca bnh DTH vi hn 10 triu heo phi git b.

4
Nm 2000, dch bnh xy ra Anh. Nm 2001, dch bnh n ra c,
Slovakia. Vo cui nm 2003, dch xut hin ti Nht, Albania v Slovakia. Hin
nay, bnh c ghi nhn khp th gii: Chu u, Trung Phi, Mexico, cc nuc
Trung m, n , Trung Quc, ng v ng Nam : Hn quc, Indonesia,
Philippine, Thi Lan, Vit Nam.
Tnh hnh bnh DTH ti Vit Nam
Ti Vit Nam, bnh DTH c Houdemer pht hin u tin vo nm 19231924. T , bnh tr thnh mi e do nghim trng i vi ngnh chn nui heo
nc ta.
Nm 1949-1950, dch bnh ln xy ra Vit Bc ri lan sang cc tnh Ph
Yn, Yn Bi, Thi Nguyn, H Ni v Hi Phng. Nm 1968-1969, dch pht ra
hn 20 tnh min Bc, theo thng k c 481 dch n ra.
Theo bo co ca Cc th y (1986), ti cc tnh Nam B bnh DTH thng
b bi nhim vi bnh ph thung hn: An Giang, Long An (1984), Tin Giang v
Hu Giang (1985). Nm 1985, ti ng Nai v TP. H Ch Minh xut hin DTH
bi nhim vi bnh t huyt trng.
Nm 2000, c 18106 trng hp bnh DTH c bo co ti Vit Nam.
Hin nay, bnh DTH vn cn tn ti cc dng bnh khng in hnh gy
tr ngi cho vic chun on.
2.1.2. Dng bnh
DTH l mt bnh truyn nhim ring ca heo. Nguyn nhn gy ra bi virus
thuc chi Pestivirus, h Flaviviridae. Virus ny c quan h mt thit vi virus gy
bnh tiu chy b (bovine viral diarrhoea-BVD) v virus gy bnh bin gii cu
(Boder disease-BD).
Bnh DTH c th xut hin nhiu dng khc nhau. Tuy nhin, ngi ta
chia bnh DTH ra lm 4 dng (Nguyn Tin Dng, 1999):

5
Dng siu cp tnh: xut hin t ngt khng c triu chng ban u, st
cao kt hp vi trng thi thng hn. Heo bnh t vong trong vng 24 n 48 gi,
cha kp xut hin triu chng trn da nn gi l DTH trng.
Dng cp tnh: gy st cao t 41 - 420C, heo bnh biu hin mt in hnh
nh heo con nm cht ng. Sau 24 - 48 gi mt sng hp v vim kt mc vi cc
triu chng ngoi da (nh vt chm tm, xut huyt, t huyt mu ti cc vng da
mng, tai, chn, bng, bao dng vt), vim d dy rut (tiu chy, nn ma),
triu chng h hp (ho, t huyt phi) v c th xut hin triu chng thn kinh
(long chong, lit, lit nh cc chi sau), t vong trong thi gian 6 n 20 ngy
sau khi pht bnh.
Dng cp tnh hoc mn tnh din ra trong 3 giai on:
Giai on u: ko di 10 - 15 ngy, ton b n heo pht bnh, cc
triu chng cc b ging nh dng cp tnh nhng mc nh.
Giai on hai l giai on thuyn gim.
Giai on ba: xut hin mm bnh bi nhim pht bnh ton thn vi
cc triu chng cc b v h hp hoc tiu ho hoc c hai (vim phi v rut thng
thng do Salmonella). Heo bnh gy mn v t vong trong vng 1 n 3 thng.
Dng khng in hnh: biu hin di cc dng rt khc nhau nh ri lon
sinh sn hoc bnh l sinh sn (sy thai, thai cht, thai g, d dng gy run ry bm
sinh, ri lon vn ng, cht yu), chm ln, cht ri rc. Di dng ny, virus
DTH c th lu hnh mt cch khng r rng, nht l heo sinh sn vi cc trng
hp lm sng l t n ra khi c cc iu kin thun li (nh stress, chuyn ch).

2.2. MT S BNH TCH C TRNG CA BNH DTH

2.2.1. DTH in hnh (cp tnh, cp tnh hoc mn tnh)
Biu hin bnh tch sung huyt hay xut huyt ca mt hay nhiu bnh ,
bin i huyt hc.

6
Trn da: xut huyt lm chm hoc tng m, chm xanh nht l da t chi
(tai, chn), xut huyt vng bng v bao quy u heo c.
Hch bch huyt: di hch vng xng chu v hch rut sng to, mu
hoa vn, t huyt hoc xut huyt vng v hoc ton b.
Thn: khng sng nhng xut huyt lm chm sau khi lt mng bao thn
(thn trng g ty).
Lch: khng sng, nhi huyt bn ra lch.
Bng quang: nim mc xut huyt lm chm.
Amian: sng to v xut huyt.
Tim: xut huyt c tim v trn vnh tim.
H h hp: t huyt, xut huyt phi, tiu thit xut huyt lm chm.
H tiu ho: t huyt v xut huyt.
Gim bch cu.
2.2.2. DTH khng in hnh
Bnh tch thay i khng c hiu: xut huyt, vim, d dng.
Da: xut huyt.
Cc hch lm ba: vim hch lm ba c th c xut huyt lm chm.
Trn no: xut huyt.

2.3. CU TRC B GEN VIRUS DTH

Virus DTH thuc chi Pestivirus, h Flaviviridae. Virus ny c chung tnh
khng nguyn vi virus bnh tiu chy b v bnh bin gii cu. Virus DTH l
mt loi virus RNA chui n dng c v bc ng knh 40 - 50 nm. RNA virus
m ho 4 protein cu trc v 7 protein khng cu trc. Ch c mt tp huyt thanh
xc nh. So vi virus gy bnh bin gii, nhng dng virus DTH hnh thnh mt
nhm khng nguyn ng dng c quan h vi nhau nhng c mt vi thay i tn
ti gia nhng dng phn lp. Nhng phn ng cho huyt thanh vi virus BVD v
BD c th xy ra v gy tr ngi trong chn on huyt thanh.

7
B gen virus c chui n RNA di 12,3 kb. B gen c bit trnh t
gen hon ton, cha mt khung c m nm bn sn ca 5-UTR v 3-UTR m
ho mt polyprotein ln vi khong 3900 amino acid. Polyprotein ny c ct bi
protease c m ho bi virus v t bo vt ch to nn protein trng thnh
ca virus gm 4 protein cu trc (C, Erns, E1 v E2), p7 v 7 protein khng cu trc
(Npro, NS2, NS3, NS4A, NS4B, NS5A v NS5B). Trnh t ca sn phm gen dc
theo khung c m l:
NH2-(Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B)-COOH

Hnh 2. 1: Cu trc b gen Pestivirus (Brett D. L., 2007)

Mt polyprotein ln c dch m t RNA ca virus s c x l thnh
nhng protein virus ring bit. Protein u tin m ho l Npro mt protein khng
cu trc c nhim v phn ct v tr Npro/C. Peptidase k ch phn ct nhng v tr
C/Erns, E1/E2, E2/p7 v p7/NS2 vi s phn ct khng hon ton v tr E2/p7.
NS2-3 c phn ct bi autoprotease NS2. S phn ct ca polyprotein hnh thnh
NS4A, NS4B, NS5A v NS5B c thu phn bi enzyme protease serine NS3-4A.
Npro l autoprotease khng cu trc c hot tnh thu phn protein. Npro
khng cn thit i vi s sao chp virus. Npro cng hot ng nh mt cht i

8
khng ca s hot ho IRF-3 v s sn xut ra IFN, c ch s phin m IRF-3
nhng t bo nhim virus DTH. Nhng t bin b i Npro ca virus DTH c
xut nh nhng d tuyn vaccin virus sng.
Protein cu trc
C (m ho protein p14): l protein ca nucleocapsid. u cui C (Cterminus) ca protein C virus DTH c xc nh v c nh v phn k
nc ca chui peptide tn hiu bn trong (internal signal peptide) khi u s di
chuyn ca Erns vo trong khoang mng li ni cht.
Erns (m ho protein gp44/48), E1(gp33), E2(gp55): l cc protein v. E2 v
Erns c tnh khng nguyn mnh nht. Erns c tc dng h tr thi virus qua mt
mng c bit, c tit ra t t bo nhim, c im ni bt ca Erns l hot tnh
ribonuclease vi tnh chuyn bit i vi gc uridine. Nhng khng th c ch hot
tnh ribonuclease c khuynh hng trung ho tnh nhim virus, s t bin Erns
ph hu hot tnh ribonuclease gy nn gia tng s lng virus. Erns ti t hp l mt
c cht i vi t bo bch huyt trong ng nghim, c th kt hp vi s gim
bch cu nhim t nhin. Mc d c tnh t bo l mt c im ni bt ca
nhng enzyme ribonuclease ho tan khc nhng ngi ta cha r l hot tnh
ribonuclease ca Erns c lin quan n c tnh ca n hay khng. Vng u cui C
(C-terminal) ca Erns c th khi ng s di chuyn ca n qua mng t bo, c th
coi nh l mc tiu hoc chc nng trong t bo. Tuy nhin, Erns ti t hp cng c
th ni mt cch vng chc vi b mt t bo qua s tng tc vi
glycosaminoglycan v c ch s ly nhim.
E1 v E2 l nhng protein mng khng th thiu. E2 ca virus DTH ti t
hp c th kt hp vi cc t bo v ngn chn s ly nhim ca virus DTH v
BVD. Mc d vai tr quan trng ca nhng glycoprotein virus l lp rp v tip
nhn nhng nhng khng th i vi Erns hoc E2 c th trung ho tnh ly nhim
ca virus v c hai khng nguyn ny c th to ra tnh sinh min dch bo v.

9
Protein p7 theo sau nhng protein cu trc, gm mt vng m ng
nhim v trung tm i vi vic tch u cui k nc v cn cho s sn sinh ca
virus ly nhim nhng khng i hi trong qu trnh sao chp RNA. P7 ca
pestivirus c phn ct mt cch khng hiu qu t E2 qua peptidase c bit. E2p7 khng phn ct khng cn thit i vi s sao chp trong nui cy t bo v c
hai E2-p7 v p7 gip t bo kt hp vi nhau. Tuy nhin, cha bit r p7 l mt
protein cu trc hay khng cu trc mc d n khng c pht hin trong virus
tinh sch. Pestivirus c p7 th c th hnh thnh nhng knh ion tham gia trong s
lp rp v tip nhn ca virus.
Protein khng cu trc
Protein NS2 l mt enzyme thu phn protein cha cysteine c nhn
din. S phn ct NS2-3 thit yu i vi s sao chp RNA ca pestivirus v hiu
qu phn ct NS2-3 c iu chnh bi mt chaperone t bo v c th xc nh
tnh gy bnh t bo. Vng NS2-3 tham gia lp rp virus.
NS3 cha mt vng protease serine u cui N v mt helicase RNA
u cui C. Protease serine NS3 i hi NS4A nh l mt yu t h tr. Protease
serine NS3-4A phn ct gia leucine v nhng amino acid khng phn cc nh.
Hot tnh protease serine nh hng n s sao chp RNA virus,ng vai tr thit
yu trong kh nng tn ti ca virus.
NS4A hot ng nh mt yu t h tr hot tnh protease serine NS3.
NS4A v NS4B khng ng vai tr thit yu trong s sao chp RNA ca virus.
NS5A v NS5B hin din di dng hai sn phm phn ct hon ton cng
khng khc g NS5A-5B khng phn ct. Chc nng ca NS5A cha c bit r.
NS5B mang c im enzyme polymerase RNA ph thuc RNA (RdRp - RNA
dependent RNA polymerase).

10

2.4. MT S PHNG PHP CHN ON BNH DTH TRONG

PHNG TH NGHIM
(1) Phn lp virus
(2) nh du min dch pht hin virus trong nui cy t bo
(3) Phng php ho m
(4) ELISA pht hin khng nguyn hoc ELISA pht hin khng th
(5) Phn ng trung ho
(6) Phng php reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR l mt phng php chn on nhanh v nhy hn so vi phng
php ELISA v phn lp virus, thch hp trong chun on bnh sm, trnh c s
nhim khun t mi trng bn ngoi
2.4.1. PCR (Polymerase chain reaction)
Khi nim: PCR l mt tin trnh lp i lp li bao gm 3 cng on: bin
tnh mu bi nhit, bt cp nhng mi nucleotide vi trnh t ch mch n, ko
di mi bt cp bi enzyme DNA polymerase chu nhit.
Bin tnh: mu DNA mch i bin tnh nhit c xc nh bi thnh
phn G+C. Thnh phn G+C cng cao th nhit i hi tch mch cng cao.
Nhng phn t DNA cng di th thi gian cn nhit bin tnh tch hai mch
mt cch hon ton cng ln. Nu nhit bin tnh qu thp hoc nu thi gian
qu ngn th ch c nhng vng giu AT s b bin tnh. Khi nhit b gim tr
hn trong chu trnh PCR, DNA mu s ti bt cp thnh mt tnh trng nguyn gc.
Trong nhng phn ng PCR xc tc bi Taq DNA polymerase, s bin tnh
c tin hnh 94 - 950C, l nhit cao nht m enzyme c th chu c
khong 30 chu k hoc hn m khng chu c tc hi qu mc. Trong chu k u
ca PCR, s bin tnh thnh thong c tin hnh khong 5 pht gia tng kh
nng nhng phn t DNA mu c bin tnh y . Tuy nhin, thi gian bin
tnh khong 45 giy nhit 94 - 950C i vi mu DNA mch thng thng
thng c thnh phn G+C l 55% hoc thp hn.

11
Mu DNA giu G+C (> 55%) th nhit bin tnh cng cao. DNA
polymerase phn lp t Archaea th chu nhit hn Taq do n thch hp khuch
i DNA giu G+C.
Bt cp mi vi DNA mu: nu nhit bt cp qu cao, mi
oligonucleotide bt cp t vi mu, nh vy lng DNA c khuch i rt thp.
Nu nhit bt cp qu thp vic bt cp ca mi khng c hiu c th xy ra,
kt qu l to ra nhng on DNA khng mong mun. S bt cp c thc hin
nhit thp hn nhit nng chy 3 - 50C. ti u nht iu kin bt cp, nn
tin hnh mt s phn ng PCR th nghim nhng nhit thp hn nhit
nng chy trong phm vi t 2 - 100C i vi hai mi oligonucleotide.
Ko di mi: vic ko di mi c tin hnh hoc gn nhit thch hp
i vi s tng hp DNA xc tc bi polymerase chu nhit, trong trng hp Taq
polymerase nhit thch hp l 72 - 780C.
S chu k: ph thuc vo lng DNA a vo phn ng, hiu qu ca vic ko
di mi v s khuch i. Thng thng sau 30 chu k to c khong 105 bn sao.
2.4.2. RT-PCR
Khi nim: l mt phng php c s dng khuch i RNA thnh
cDNA. Nhy v a nng, RT-PCR c s dng hi phc v nhn u cui 5v
3 ca mRNA v hnh thnh th vin cDNA t mt lng nh mRNA. Thm vo
, RT-PCR d dng xc nh t bin v nhng a hnh trong nhng trnh t c
sao li v o lng nng biu hin gen khi lng RNA b hn ch.
Bc u tin l chuyn RNA thnh cDNA. Mt mi oligodeoxynucleotide
c lai vi RNA v sau ko di bi polymerase DNA ph thuc RNA (RNAdependent DNA polimerase) to ra bn sao cDNA. Kt thc tin trnh to cDNA
l chuyn qua tin trnh PCR nhn ln mt lng ln cDNA.

12
Khun RNA

S gn primer vo RNA tng hp cDNA

(primer c th l ngu nhin, oligo-dT hay
mi chuyn bit cho gene).

cDNA c tng hp bt u t v tr ca primer nh

enzyme reverse trascriptase.

Si cDNA c to thnh.

Khun RNA c loi b nh Rnase H.

cDNA c s dng thc hin PCR.

S gn ca primer vi cDNA.

Si b sung ca cDNA c tng hp nh

Taq polymerase.

cDNA si i c hnh thnh.

Ba bc bin tnh, bt cp, ko di c lp

li nhiu ln.

Hnh 2. 2: Phn ng RT-PCR

.

13
2.4.3. Cc thnh phn quan trng trong phn ng
Dung dch m (buffer)
C tc dng to ra lc ion cn thit cho phn ng xy ra. Thnh phn dung
dch m gm KCl, MgCl2, Tris-HCl (pH 8,5 nhit phng).
MgCl2
To thnh mt phc hp vi dNTP cn thit cho vic gn dNTP vo
enzyme, kch thch hot tnh ca enzyme polymerase, tng Tm (nhit nng chy)
ca DNA v tng s tc ng h tr ca mi v DNA mu.
Nng cui MgCl2 trong phn ng thng trong khong 0,5 5 mM vi
mc ti u l 1 2 mM, nhng nng ti u phi c xc nh cho tng phn
ng qua nhiu th nghim (H Hunh Thu Dng, 1998).
Mi (primer)
Mi l mt trong nhng thnh phn quan trng nht nh hng trc tip n
hiu qu cng nh chuyn bit ca phn ng.
Trnh t mi c chn sao cho khng c s bt cp b sung gia hai mi
v c trng cho trnh t on gen c khuch i, khng trng vi cc trnh t
lp li trn on gen. Chiu di mi khng c qu ln thng trong khong 1725 nu, phn ng PCR thng ti u vi nhng on trnh t nh hn 1kb (Phan C
Nhn, 2001). Chiu di mi cng ln s tch cc DNA mu bt cp vi mi cng
nh. Chiu di v thnh phn G-C ca mi u c nh hng quan trng i vi
nhit Tm ca mi.
Tm (0C) = [(s G + C) * 4 + (s A + T) * 2]
Nng ca hai mi cng nh hng ln n phn ng. Nng mi qu
cao hnh thnh nn cu trc dimer (mi gn mi).
Deoxyribonucleotide triphosphates (dNTPs)
L nguyn liu cn thit cho phn ng gm 4 loi: dATP, dTTP, dCTP,
dGTP. S mt cn bng trong thnh phn dNTPs lm tng cc li sao chp ca

14
enzyme polymerase v vy phi gi cho nng ca tt c cc dNTP u bng
nhau. Nng dNTPs thng dng trong khong 20 200 M, dung dch dNTPs
gc phi gi trung tnh pH7,0.
Taq polymerase
L mt enzyme polymerase chu nhit, c tch chit t vi khun sui
nc nng c tn l Thermus aquaticus. Taq polymerase khng b ph hu nhit
bin tnh trong phn ng, hot ng ti u nhit 72 - 780C.
Nng Taq s dng c khuyn co trong khong 1 - 2,5 n v trn
100l dung dch phn ng. Nng Taq qu cao s to ra nhng sn phm khng
chuyn bit, nng Taq qu thp khng lng enzyme xc tc to ra sn
phm theo mong mun (Bi Ch Bu, 1999).
S chu k trong phn ng
S chu k cho mi phn ng ph thuc vo lng bn sao mu ban u.
DNA mu l 105 tng ng vi 25 - 30 chu k, 102 - 103 tng ng vi 35 - 40 chu
k.
Khng nn vt qu 40 chu k v hiu qu khuch i s gim dn do:
S phn hu v cn kit cc thnh phn ca phn ng.
S xut hin cc sn phm ph c ch phn ng.
Cc bn sao va tng hp khng kt hp vi mi m bt cp vi nhau
(Phan C Nhn, 2001).
Mu
Nng thng bin ng trong khong 1 pg 1 g trn 25 l dung dch
phn ng. Nng mu qu t dn n phn ng khng c hiu, nng qu cao
to ra nhng sn phm ph khng mong mun.
RNAsin
c dng c ch enzyme phn hu RNA, gi cho mu khng b mt
trong qu trnh thc hin phn ng.

15
Enzyme reverse transcriptase
Trong phn ng RT-PCR thnh phn khng th thiu l enzyme phin m
ngc, MMLV hot ng nh l mt enzyme polymerase gip tng hp si cDNA
t RNA v vi hot tnh ca enzyme RNAse H c nhy cao gip phn ct si
RNA trong mch i RNA-cDNA to ra mch n cDNA tip tc cho phn ng
PCR.

2.5. S LC CC NGHIN CU LIN QUAN

2.5.1. Trong nc
T nm 1996-1998, Nguyn Th Phng Duyn v ctv kho st hi chng
st, to bn, b n n cht v khng nguyn virus DTH bng min dch hunh
quang v ELISA n heo sinh sn v heo con tnh Khnh Ho v Qung Ngi cho
thy virus DTH chim t l khng nh trong cc tc nhn gy hi chng ny.
T nm 2002-2003, Akemi Kamakawa v ctv tin hnh kho st bnh DTH
gia cc n heo v cc tri heo ti Cn Th bng k thut RT-PCR khuch i
on gen 5NTR.
Nguyn Th Vinh v ctv, nm 2004 nghin cu phn tch di truyn virus
DTH phn lp Vit Nam bng cch phn tch on gen E2 ca 20 mu virus DTH
v so snh chng vi mt s chng c gii thiu trn th gii. Kt qu a ra
l cc chng virus DTH thuc nhm 2 ang l tc nhn chnh gy bnh DTH Vit
Nam.
H Thu Hng v ctv, nm 2004 so snh 4 phng php chn on virus
DTH (phn lp virus, phn ng hunh quang khng th, phn ng ELISA v RTPCR) t mu c bo qun cc iu kin khc nhau. Theo cc tc gi, vic la
chn phng php chn on phi da vo cht lng mu.
Bi Ngha Vng v ctv, nm 2004 chn on bnh DTH bng phng
php RT-PCR vi mu mu ly bng giy thm chng t rng c th pht hin c
virus DTH t cc mu giy thm mu kh gi 370C trong 10 thng.

16
2.5.2. Trn th gii
Vi s thit hi kinh t nghim trng m bnh DTH gy ra cc quc gia c
dch bnh th DTH l mt i tng ngy cng c nhiu nghin cu v dch t lm
sng, cc phng php chn on, s ly nhim v phn b ca cc chng virus
DTH v ng thi nghin cu vaccin trong phng tr bnh.
Barbara v ctv, nm 1993 s dng k thut RT-PCR vo vic pht hin v
bit ho virus DTH t cc pestivirus khc.
Nghin cu du hiu lm sng v dch t ca bnh DTH ca cc tc gi nh
Artois v ctv (2002), Moennig v ctv (2003).
Nhiu tc gi nc ngoi dng k thut RT-PCR nghin cu c im
dch t bnh da trn on gen E2 ca b gen virus (Wirs B., 1993; Harding, 1994;
Rugg li,1996; Knepp, 2003; Risatt I, 2002, 2004; Pereda, 2005).
Paton v ctv (2000) phn tch cc chng virus DTH da vo on gen E2,
NS5B v 3NTR nhiu ni trn th gii, k c vi nc chu kt qu cho thy
mi quan h gn gia cc chng gy bnh mt s nc.

17

Chng 3
NI DUNG V PHNG PHP TIN HNH
3.1. THI GIAN V A IM
3.1.1. Thi gian
ti c tin hnh t ngy 01 thng 03 nm 2007 n ngy 01 thng 08
nm 2007.
3.1.2. a im
Mu bnh phm l lch ca heo bnh v cc d liu lin quan n bnh
c gi t tnh Tin Giang.
Tin hnh th nghim quy trnh pht hin virus DTH da trn on gen
NS5B ti Trung tm Phn tch Th nghim Ho sinh trng i hc Nng Lm
thnh ph H Ch Minh.

3.2. NI DUNG NGHIN CU

Chn quy trnh ly trch RNA thch hp t mu lch
Chn quy trnh RT-PCR c hiu qu nht trong vic pht hin gen NS5B
So snh kt qu RT-PCR vi kt qu OD ca ELISA
Kho st mi lin h gia kt qu RT-PCR vi c im bnh tch.

3.3. VT LIU V HO CHT

3.3.1. Vt liu nghin cu
Mu dng trong ly trch RNA (lch c xt nghim ELISA dng tnh
vi khng nguyn E2 c gi t Chi cc Th y Tin Giang, cc mu dng tnh
khi t s OD t 0,3 tr ln) v phn ng RT-PCR pht hin gen NS5B.

18
3.3.2. Ha cht
Ho cht dng trong ly trch RNA tng s
Dung dch D l dung dch ly gii t bo:
2 M guanidium thiocyanate
25 mM sodium citrate
0,5% sarkosyl (w/v)
100 mM -mercaptoethanol, pH7
2 M sodium acetate, pH4
Phenol
Chloroform
Isopropanol
Ethanol 75%
Ho cht dng trong in di gel bin tnh
Dung dch m: 10X MOPS electrophoresis buffer (500 ml), thnh phn:
0,2 M MOPS (pH7)
20 mM sodium acetate
10 mM EDTA
Dung dch np mu: 10X formaldehyde gel-loading buffer (10 ml), thnh
phn:
50% glycerol
10mM EDTA
0,25% (w/v) bromophenol blue
0,25% (w/v) xylene cyanol
Ho cht v dng c trong in di gel TBE
Dung dch m TBE 50X
Tris

242 g/l

Boric acid

57.1 ml/l

EDTA 0.5M pH 8.0

100 ml/l

19
Agarose (Biorad)
Dung dch np mu (loading dye) 10X (20% Ficoll 400; 0,1 M disodium
EDTA, pH 8; 1% sodium dodecyl sulfate; 0,25% bromphenol blue; 0,25%
cylene cyanol)
Dung dch ethidium bromide (dung dch stock 1000X 0,5 mg/ml: 50 mg
ethidium bromide, 100 ml H2O. Dung dch s dng 0,5 g/ml pha long
1:1000 cho gel hoc dung dch nhum bo qun trnh nh sng).
Thang DNA chun
B dng c in di nm
B ngun in mt chiu
Lc gel
Khun gel
Micropipette cc loi.
Ho cht dng trong phn ng RT-PCR
Rnase-free water
Buffer PCR
MgCl2
dNTPs
Mi xui
Mi ngc
Taq polymerase
Rnasin
MMLV reverse trancriptase
Triton X100

3.4. B TR TH NGHIM
Th nghim 1: Kt ta RNA ly trch cc nng LiCl khc nhau
LiCl l mt tc nhn kh nc mnh, vi c tnh ho tan RNA thp hn
DNA do mui LiCl nng cao c s dng kt ta RNA (Sambrook v

20
Russell, 2001). kho st hiu qu kt ta ca mui LiCl vi RNA, trong qu
trnh ly trch mu chng ti thc hin thu ta RNA vi cc nng mui LiCl khc
nhau. T chn nng mui LiCl thch hp nht trong thu ta RNA, a ra quy
trnh tch chit RNA thch hp.
Th nghim ny c b tr theo kiu hon ton ngu nhin. Gm 4 nghim
thc (LiCl 1,0 M; LiCl 1,5 M; LiCl 2,0 M; LiCl 2,5 M), mi nghim thc c lp
li trn 5 mu.
Ch tiu kho st l t s OD, hm lng RNA thu c v sn phm RTPCR thu c t cc mu. So snh s khc bit gia cc nghim thc, chn ra
nghim thc thch hp nht.
Th nghim 2: Th nghim hm lng Taq khc nhau trong phn ng
RT-PCR
Hm lng Taq c khuyn co trong khong 1 2,5 UI trn 100 l dung
dch phn ng. Nng Taq qu cao s to ra nhng sn phm khng chuyn bit
(Bi Ch Bu, 1999). V vy, bc u chng ti thc hin th nghim thay i
lng nh nng Taq trong phn ng.
Th nghim c thc hin trn 5 mu khc nhau, mi mu c chy phn
ng RT-PCR vi 2 nng Taq khc nhau l 2,0 UI v 2,5 UI.
Ch tiu kho st th nghim ny l t l mu thc hin phn ng thnh cng
v bng sn phm RT-PCR c in di trn gel 2%.
Kho st 3: So snh kt qu OD ca ELISA vi kt qu RT-PCR
Nhm kho st c hay khng s ph thuc kt qu RT-PCR vi t s OD
ca ELISA
Vi 23 mu bnh phm c kt qu OD ca ELISA pht hin khng nguyn
E2 c gi t Chi cc Th y Tin Giang so snh vi kt qu RT-PCR chng ti
thc hin ti Trung Tm Phn Tch Th Nghim Ho Sinh trng i hc Nng
Lm.

21
Kho st 4: Kho st mi lin h gia c im bnh tch ca nhng
heo chn on dng tnh vi bnh DTH bng phng php RT-PCR
Bnh DTH vi cc triu chng lm sng, bnh tch khng u nhau v c
nhiu bin i khng in hnh. V vy, chng ti xem xt nhng mu dng tnh
trong phng php chn on RT-PCR c nhng c im bnh tch nh th no.
Kho st c thc hin da trn kt qu RT-PCR khuch i gen NS5B
23 mu lch vi nhng c im bnh tch nghi ng bnh DTH (lch s mu c
gi t Chi cc Th Y Tin Giang).

3.5. PHNG PHP TIN HNH

3.5.1. Thu thp mu bnh phm
Ly mu khi heo c mt s bnh tch nghi ng ca DTH nh sau:
Da xut huyt
Gan xut huyt, hoi t
Lch xut huyt, nhi huyt hoc hoi t ra lch
Thn xut huyt, xung huyt
Phi xut huyt, t huyt, hoi t hoc phi ho gan (mu sc ging
gan, th vo nc b chm)
Tim xut huyt
D dy xut huyt
Rut xut huyt hnh cc o
Mu bnh phm l lch c xt nghim ELISA pht hin khng nguyn
E2 dng tnh.
Ly khong 100 g mu cho vo ti nilng v trng c bo qun trong
bnh v vn chuyn v phng th nghim, bo qun nhit -700C ti Trung
Tm Phn Tch Th Nghim Ho Sinh trng i Hc Nng Lm Thnh ph H
Ch Minh cho n khi xt nghim.

22
3.5.2. Ly trch RNA tng s
Mu c th c x l theo 2 cch:
Mu bnh phm lch c nghin trong dch PBS thu huyn dch t bo
(t l mu so vi dch PBS l 20%)
Cc bc tin hnh:
Bc 1: Ct mt phn lch cho vo ci, dng ko ct nh, cho mt lng
dch PBS tng ng vo, dng chy nghin nhuyn cho n khi khng cn cn.
Bc 2: Thu dch nghin vo eppendorf ln, gii ng 3 ln.
Bc 3: Ly tm 5000 vng/10 pht 40C
Bc 4: Thu dch t bo, b cn. Dch t bo thu c bo qun -700C
cho n khi tin hnh ly trch mu.
Mu bnh phm nghin trong nit lng -1960C thu mu dng bt nhuyn.
Cc bc tin hnh:
Bc 1: Ci chy c lm lnh vi nit lng -1960C, ct mt lng mu
cho vo ci chy, vo mt lng nh nit, dng chy nghin nhuyn.
Bc 2: Khi mu dng bt nhuyn, thu mu vi lng khong 30 mg
cho vo mi eppendorf. Mu c bo qun -700C cho n khi s dng.
Tin hnh ly trch RNA tng s
Nguyn tc ly trch RNA t mu dch t bo da theo quy trnh ca
Chomezynski v Sacchi (1987):
Dch t bo c ng nht chung vi dung dch D v -mercaptoethanol
nhm ph v mng t bo phng thch ra cc thnh phn ni bo (DNA, RNA,
protein). ng thi guanidium thiocyanate c trong dung dch D lm bin tnh
protein mnh v c ch hot ng ca Rnase.
Phenol, chloroform cho vo tip sau nhm kt ta protein v pH4,0 ca
phenol c tc dng ko DNA vo pha phenol. Ly tm tc cao nhm thu c
dch trong bn trn cha RNA, nhit lnh hn ch s phn hu RNA.

23
Dch trong thu c vi ethanol b sung LiCl nhm kt ta c hiu RNA.
LiCl l mt tc nhn kh nc mnh, vi c tnh ho tan RNA thp hn DNA do
c dng kt ta RNA vi nng cao LiCl (Sambrook v Russell, 2001).
Ta RNA c ra vi ethanol 75% t 2-3 ln nhm loi b LiCl, gim
nng ethanol ban u. Ta RNA sau khi c lm kh th ho tan trong nc
c kh vi DEPC v bo qun -700C nhm trnh s phn hu ca Rnase.
Cc bc tin hnh ly trch:
Ht khong 350 l dch t bo vo ng eppendorf.
Cho vo 500 l dung dch D v 3,6 l -mercaptoethanol.
ng nht mu, vortex trong 10 giy.
Sau cho thm 50 l sodium acetate 2 M, pH 4,0; 500 l phenol bo
ho pH4,0; 100 l chloroform.
Dng micropipet trn u ng nht mu, vortex 10 giy.
Lm lnh trn khong 15 pht.
Li tm 13000 vng/pht trong 20 pht 40C.
Ht khong 400 l dch ni, thm 400 l ethanol + LiCl v gi -200C
trong 1 gi.
Li tm 13000 vng/pht trong 20 pht, b dch ni, thu ta.
Ra ta bng ethanol 75%, votex nh, li tm 9000 vng/pht trong 2 pht,
thu ta (ra 2 ln).
Ht b dch ni, ta lm kh t nhin trong khng kh khong 45 pht.
Ho tan vi 30 l nc kh DEPC.
nh lng RNA bng quang ph k
o tinh sch v hm lng RNA sau khi ly trch bng quang ph k
(model HP 8453) bc sng 230 nm, 260 nm v 280 nm
Cch tin hnh: Curvet c ra sch 2 ln vi nc kh DEPC. Ht 995
l nc kh DEPC cho vo curvet, sau thm 5l mu trn u pha
long 200 ln. Cui cng l t curvet vo my o OD.

24
tinh sch ca RNA c tnh bng t s OD260nm /OD280nm v t s
OD260nm /OD230nm . T s OD260nm/OD280nm t 1,8-2,0 th xem nh RNA ly trch
tng i sch. T s ny s thp hn khi nhim protein hay phenol. T s
OD260nm/OD230nm t 1,5-2,0 coi nh RNA t tp nhim, t s ny thp hn khi b
nhim guanidium thicyocyanate trong nhng bc kt ta.
Hm lng RNA c tnh theo cng thc:
Hm lng RNA (ng/l) = WL1 (OD260nm)* 40 ng/l* pha long
Mt n v OD260nm tng ng vi mt nng 40 ng/l cho mt dung dch
RNA hay DNA si n (H Hunh Thu Dng, 1998)
RNA ly trch c th dng thc hin tip phn ng RT-PCR.
Kim tra RNA bng in di bin tnh:
Xem kt qu c xut hin 2 band chun 18S, 28S.
Nu kt qu c xut hin 2 band 18S v 28S th qu trnh ly trch RNA thnh cng.
Tin hnh in di:
Chun b gel 1,5%:
Cn 0,3 g agarose, thm vo 14,4 ml nc kh DEPC.
Nu trong 2 pht 650W
ngui n 550C, cho thm vo:
2 ml MOPS 10X
3,6 ml formaldehyde
Lc u, gel v ch gel ngui khong 45 pht.
Chun b RNA:
Mi phn ng cn:
10X MOPS

2 l

Formaldehyde

4 l

Formamide

10 l

RNA

10 l

650C trong 15 pht.

15 pht.

25
Cho thm 2 l loadingdye trn u v bm vo ging trn gel.
in di 30V trong 180 pht hoc 50V trong 90 pht.
Sau ly gel ra em nhum ethium bromide trong 30 pht, chp
Cui cng l chp gel v c kt qu.
3.5.3. Phn ng RT-PCR
Quy trnh RT-PCR c thc hin theo dn liu ca Paton v ctv (2000).
Cp mi ca gen NS5B:
Forward: 5-GAC ACT AG(TC) GCA GGC AA (TC) AG-3 (11138-11157)
Reverse: 5-AGT GGG TTC CAG GA(GA) TAC AT-3 (11586-11567)
Kch thc on gen l 449 bp.
Cp mi ny s dng pht hin phm vi rng nhng chng virus DTH phn
lp (Paton, 2000). Nhng th nghim da trn s khuch i gen NS5B c s
dng rng ri trong vic phn loi di truyn, do phng php RT-PCR da trn
on gen NS5B thch hp l phng php chn on tiu chun ho i su xc
nh chnh xc chng virus DTH.
Quy trnh RT-PCR: chun b 50 l hn hp phn ng RT-PCR vi cc thnh phn
phn ng nh sau:
Bng 3. 1: Thnh phn phn ng RT-PCR
Tn ho cht
Rnase- free water
Buffer PCR
MgCl2
dNTPs
Mi xui
Mi ngc
Triton X-100
Taq polymerase
Rnasin
MMLreverse trancriptase
RNA mu
Tng th tch

Nng cui
1X
6 mM
0,4 mM
0,1 M
0,1 M
0,2%
2,5 UI
10UI
100 UI
50l

26
Buffer 10X gm: 500 mM KCl, 100 mM Tris-Cl (pH 8,3 nhit phng), 15 mM
MgCl2
Quy trnh nhit i vi gen NS5B:
Giai on RT:

500C/30 pht, 950C/3pht.

Giai on PCR:

35 chu k (940C/1pht, 520C/1pht,720C/1pht)

Bc ko di chui:

720C/10pht

in di trn gel
Cch tin hnh:
Pha long dung dch TBE 50X c dung dch TBE 0,5X.
Pha gel agarose vi nng 2%: Cn 0,25 g agarose cho vo 12,5 ml
dung dch TBE 0,5X. un si bng l viba cho agarose tan tht u.
ngui n 50-550C, vo khun, ci lc vo.
Ch 30 pht agarose ng.
G lc ra ri t bng gel vo bun in di cho ng chiu. Cho dung
dch TBE ngp gel.
Load mu vo cc ging vi t l 2 l loading dye v 6 l mu.
Chy in di iu kin 100 V, 400 mA trong 20 pht hoc 60 V, 250
mA, thi gian khong 60 pht nu chy chung vi thang chun.
Nhum ethidium bromide khong 20 pht.
Gel sau khi nhum s c chp bng tia t ngoi UV. Nu mu c sn
phm th bng sn phm s pht sng di dng vch trn gel in di. m
nht ca bng in di phn nh nng cao hay thp ca sn phm RTPCR thu c.
c kt qu in di
Gen NS5B vi sn phm thu c l bng in di c kch thc khong 449 bp.

3.6. X L S LIU
S liu dng lin tc c phn tch bng trc nghim F. So snh t l bng
Chi bnh phng.

27

Chng 4
KT QU V THO LUN
4.1. KT TA RNA CC NNG LICL KHC NHAU
Chng ti thc hin ly trch RNA t mu lch da theo quy trnh ca
Chomeszynski v Sacchi (1987) v thu ta RNA bng ethanol 100% kt hp vi
LiCl cc nng 1,0 M; 1,5 M; 2,0 M; 2,5 M. Kt qu thu c bng 4.1.
Bng 4. 1: T s OD v hm lng RNA cc nng LiCl
Nng LiCl

Ch tiu

1,0 M

T s OD
Hm lng RNA
(g/l)

2,5 M

1,89 0,2 1,99 0,2

2,08 0,2

2,46 0,2

0,23

1,45 0,2 1,61 0,2

1,29 0,2

1,07 0,2

0,2

5
2,46

2,5
2

2,0 M

S mu

1,5 M

1,99

1,89
1,45

1,5

2,08

1,61
1,29

1,07

T s OD
Hm lng RNA (g/l)

0,5
0
1M

1,5M

2M

2,5M

Biu 4.1: T s OD v hm lng RNA cc nng LiCl

Kt qu bng 4.1 cho thy tinh sch v hm lng RNA thu c cc
nng LiCl khc nhau khng c s khc bit c ngha v phng din thng k

28
(P > 0,05). Tuy nhin, s khc bit th nghim ny l sn phm RT-PCR thu
c, th hin hnh 4.1.

500 bp

449 bp

Hnh 4.1: Sn phm RT-PCR ca mu c kt ta cc nng LiCl

Ch thch:
L: Thang chun (100 bp)
1: Kt ta RNA bng ethanol 100%
2: Kt ta RNA bng ethanol 100% + LiCl 1,0 M
3: Kt ta RNA bng ethanol 100% + LiCl 1,5 M
4: Kt ta RNA bng ethanol 100% + LiCl 2,0 M
5: Kt ta RNA bng ethanol 100% + LiCl 2,5 M
T kt qu thu c chng ti nhn xt rng sn phm khuch i cc
nng LiCl khc nhau th khc nhau, bng sn phm in di thu c r dn v
lng tp cht gim dn theo nng LiCl tng dn. iu ny c th c gii
thch do cht lng RNA ly trch. T hnh 4.1 nhn thy sn phm ging th 5
(LiCl 2,5 M) r nht v tp t nht v t biu 4.1 nhn thy tinh sch mu cao
nht nng LiCl 2,5 M. iu ny c th gii thch RNA thu c nng
LiCl cao t ln tp hn. V vy chng ti chn nng LiCl 2,5 M kt ta RNA
trong qu trnh ly trch mu.

29
4.2. THAY I NNG TAQ TRONG PHN NG
th nghim ny chng ti thc hin phn ng RT-PCR vi 2 nng Taq
khc nhau l 2,0 UI v 2,5 UI, mi nng c thc hin 5 phn ng. Kt qu thu
c bng 4.2.
Bng 4.2: Kt qu RT-PCR 2 nng Taq
Kt qu RT-PCR

Nng Taq

S mu thc hin

S mu thnh cng

T l thnh cng

2,0 UI

100%

2,5 UI

100%

Kt qu in di sn phm RT-PCR thu c 2 nng Taq.

Ch thch:
L: Thang chun (100 bp)
1: nng Taq 2,0 UI

449 bp

2: nng Taq 2,5 UI

Hnh 4.2: Sn phm RT-PCR 2 nng Taq

T kt qu bng 4.2 v hnh 4.2 cho thy khng c s khc bit gia 2 phn
ng 2 nng Taq 2,0 UI v 2,5 UI. V vy, chng ti chn phn ng RT-PCR
vi nng Taq 2,0 UI, gim c lng Taq phn ng m cht lng phn ng
vn khng thay i, gim c chi ph phn ng.

30
4.3. SO SNH T S CA ELISA VI KT QU RT-PCR
Bng 4.3: T s OD ca ELISA v kt qu RT-PCR
S th t

K hiu mu

Kt qu OD ca ELISA

Kt qu RT-PCR

20

1,399

22

0,596

23

0,613

24

1,656

25

0,617

26

1,579

27

0,641

29

2,248

30

3,535

10

33

3,268

11

37

3,327

12

43

2,751

13

45

1,114

14

53

0,953

15

54

0,928

16

57

2,746

17

60

3,471

18

0,492

19

11

1,006

20

13

0,726

21

14

0,492

22

16

0,727

23

17

1,307

31
Chng ti so snh kt qu RT-PCR vi 3 khong t s OD ca ELISA l t
0,49 1; > 1 - 2 v > 2 3,5. Kt qu so snh c trnh by bng 4.4.
Bng 4.4: So snh cc mc OD ca ELISA v RT-PCR
T S OD CA ELISA

RT-PCR (+)

RT-PCR (-)

TNG

T L (+)

0,49 1

10

11

9%

>12

20%

> 2 3,5

71%

TNG

16

23

30,4%

SAI BIT THNG K

P = 0,018

T kt qu bng 4.4 cho thy kt qu RT-PCR ph thuc vo t s OD ca

ELISA (P < 0,05). T l (%) mu dng tnh vi RT-PCR cao (71%) tng ng vi
mc t s OD > 2 3,5. iu ny c th gii thch hm lng khng nguyn c
trong mu xt nghim t l thun vi t s OD ca ELISA. Hu ht cc khong t
s OD u c mu dng tnh vi RT-PCR; tuy nhin, t l (%) mu dng tnh vi
RT-PCR so vi tng s mu dng tnh vi ELISA khng cao. iu ny mt mt
c th do nh hng yu t OD trong ELISA; mt khc do nh hng cc yu t bt
cp trong qu trnh ly trch RNA (giai on vortex, giai on phi mu), bo qun
mu cng nh cc yu t bt li ca mi trng lm vic c th tc ng n s
phn hy RNA trc khi bc vo giai on RT-PCR.

32
4.4. MI LIN H GIA KT QU RT-PCR VI C IM BNH TCH
Bng 4. 5: c im bnh tch cc mu RT-PCR dng tnh v m tnh
C quan

Bnh tch

RT-PCR(+)

RT-PCR (-)

T l (%

T l (%)

Da

Xut huyt

100

16

100

Thn

Xut huyt
Sung huyt
Hoi t im
Vim dnh
Lot cc o
Khng biu hin

3
1
0
0
1
2

42,8
14,3
0
0
14,3
28,6

2
2
3
1
0
8

12,5
12,5
18,75
6,25
0
50

Rut gi

Xut huyt
Lot cc o
Khng biu hin

4
1
2

57,1
14,3
28,6

5
1
10

31,25
6,25
62,5

Hch
mng
treo rut

Xut huyt
Sng
Sng + xut huyt
Sng + t huyt
Khng biu hin

3
0
3
0
1

42,8
0
42,8
0
14,3

3
1
0
1
11

18,75
6,25
0
6,25
68,75

Bng
quang

Xut huyt
Khng biu hin

3
4

42,8
57,9

1
15

6,25
93,75

Lch

Xut huyt
Nhi huyt
Nhi huyt + xut huyt
Sng+XH
Sng+NH
Khng biu hin

1
5
0
1
0
0

14,3
71,4
0
14,3
0
0

4
9
1
0
1
1

25
56,25
6,25
0
6,25
6,25

Van hi
manh
trng

Xut huyt
Khng biu hin

4
3

57,9
42,1

2
14

12,5
87,5

33
T kt qu bng 4.6 cho thy cc mu dng tnh vi RT-PCR u c biu
hin bnh tch xut huyt hu ht cc c quan (> 42%) so vi cc bnh tch khc,
tuy nhin lch biu hin bnh tch nhi huyt chim t l cao hn (71,4%). ng
thi cc mu RT-PCR m tnh biu hin bnh tch nhi huyt lch cng chim t
l cao 56,25% nhng cc bnh tch khc ri rc chim t l khng cao. V vy,
chng ti nhn xt rng bnh DTH thng biu hin bnh tch xut huyt hu ht
cc c quan, kt hp vi nhi huyt lch cng vi nhng biu hin bnh tch
khng u nh sng, sung huyt, lot cc o cc c quan.
Thng thng, chun cao virus c quan st trong lch, xng, nhng
hch bch huyt ni tng v nhng cu trc dng bch huyt mng treo rut non
(Artois v ctv, 2002). Theo quan st trong khi lm th nghim hu ht cc mu bnh
phm dng tnh l mu lch c nhi huyt quanh ra lch. V vy, chng ti cho
rng mu lch thch hp dng lm mu bnh phm trong chn on DTH. Ngoi ra,
t bng 4.6 cho thy hch mng treo rut biu hin xut huyt t l cao (85,6%)
cc mu chn on dng tnh; trong khi cc mu m tnh t l thp hn (25%).
Do , chng ti nhn xt rng ngoi mu lch th hch mng treo rut cng c
xem l mu thch hp dng trong chun on bnh DTH.

Ch thch:
L: Thang chun (100 bp)
C: i chng (+) (cung cp bi
Trung tm Th y vng thnh
ph H Ch Minh)
1, 2, 3, 4: Cc mu c virus DTH

Hnh 4. 3: Kt qu sn phm RT-PCR cc mu bnh phm

34

Chng 5
KT LUN V NGH
5.1. KT LUN
(1) Xc nh c quy trnh tch chit RNA thch hp t mu lch. Nng
LiCl 2,5 M cho ta RNA vi tinh sch v cht lng sn phm khuch
i cao.
(2) a ra c quy trnh RT-PCR mt bc pht hin gen NS5B ca virus
DTH vi nng Taq gim t 2,5 UI xung 2 UI nhng cht lng sn
phm khuch i khng thay i.
(3) Mc t s OD ca ELISA ca cc mu ln hn 2 cho kt qu RT-PCR
dng tnh cao.
(4) Kt hp c nhng c im bnh tch thng gp heo nhim bnh DTH
thng qua kt qu chn on RT-PCR dng tnh(bnh tch xut huyt
hu ht cc c quan kt hp vi nhi huyt lch cng vi mt s biu hin
bnh tch khc)

5.2. NGH
(1) Thc hin thm nhiu th nghim trong qu trnh ly trch cng nh trong
phn ng RT-PCR pht hin virus nhm to ra c mt quy trnh chn on
ti u nht ng dng trong thc tin chn on bnh DTH nhanh, t tn km
nht.
(2) Tin hnh xc nh trnh t nucleotide ca sn phm khuch i khng
nh chnh xc virus DTH. ng thi so snh trnh t ca virus phn lp
c vi trnh t ca cc chng virus phn lp c trong d liu NCBI
nhm nh chng virus DTH hin nay nc ta.

35

TI LIU THAM KHO

Ti liu ting Vit
1. Bi Ch Bu v Nguyn Th Lang, 1999. Di truyn phn t - Nhng
nguyn tc c bn trong chn ging cy trng. Nh xut bn Nng
Nghip, trang 195 209.
2. Bi Ngha Vng, Ken Inui, H Thu Hng, Nguyn Tin Dng. Chun
on bnh dch t heo bng phng php RT-PCR vi mu mu ly bng
giy thm. Bo co khoa hc Chn nui Th y 2002-2003.
3. H Hunh Thu Dng, 2002. Sinh Hc Phn T (Khi nim Phng
php - ng dng). Ti bn ln 2, NXB Gio Dc, Thnh Ph H Ch
Minh.
4. H Thu Hng , Ken Inui, Bi Trng Ngha, o Thanh Vn, Nguyn
Thu Duyn, Kenji Kawashima v Nguyn Tin Dng, 2004. So snh 4
phng php chun on virut dch t heo t mu c bo qun cc
iu kin khc nhau. Bo co khoa hc CNTY, B NN & PTNT, 13-16.
5. Kim Vn Phc, ng Hng, Phm Th Vui, Chris Morrissy, Nguyn Th
Lam Hng, Nguyn Th Thu Hng, Trn nh T, 2004. Nghin cu ng
dng k thut RT-PCR pht hin virus dch t heo. Bo co khoa hc
Chn nui Th y, B NN & PTNT, 360-367.
6. Nguyn Th Vinh, Ken Inui, H Thu Hng v Nguyn Tin Dng, 2004.
Phn tch di truyn virus dch t ln Vit Nam. Bo co khoa hc Chn
nui Th y, B NN& PTNT, 10-13.
7. Nguyn Th Phng Duyn, Vn Khuyn, D nh Qun, 2001. Kho
st hi chng st, to bn, b n n cht n ln sinh sn v n ln
con tnh Khnh Ho v Qung Ngi. Bo co khoa hc CNTY, B NN &
PTNT, 10-12/4/2001.

36
8. Nguyn Tin Dng, 1999. Dch t ln c in lun l vn thi s: Tnh
hnh hin ti v bnh ng s ny. Tp ch khoa hc k thut Th y, tp 4,
2:1999.
9. Nguyn Tin Dng, Nguyn Vn Quang, H Thu Hng, Ng Thanh
Long, o Thanh Vn. Tnh hnh nhim bnh virut trong n tru b Vit
Nam. Bo co khoa hc Chn nui Th y 2002-2003.
10. Phan C Nhn, 2001. Di truyn hc ng vt. Nh xut bn Khoa Hc v
K Thut H Ni.

Ti liu ting Anh

11. Akemi Kamakawa, Ho Thi Viet Thu, Shunji Yamada, Masanori Kubo,
Seishi Yamasaki v Toshiaki Taniguchi, 2003. Classical swine fever
among pig herds and its control in Cantho province, Mekong delta.
Department of Veterinary Medicine, Faculty of Agriculture, Can Tho
University.
12. Artois M., K.R. Depner, V. Guberti, J. Hars v S. Rossi, 2002. Classical
swine fever (hog cholera) in wild boar in Europe. Rev. sci. tech. Off. Int.
Epiz., 2002, 21 (2), 287-303.
13. Brett D. Lindenbach, Heinz-Jurgen Thiel v Charles M. R, 2007.
Flaviviridae: The viruses and their replication. Fields Virology, 5th
Edition. D. M. Knipe and P. M. Howley, Eds. Lippincott-Raven
Publishers, Philadelphia (2007).
14. Chomczynski and Sacchi, 1987. Single-step method of RNA isolation by
acid guanidinium thiocyanate - phenol - chloroform extraction. Analytical
Biochemistry 162: 156-159
15. EU diagnostic manual for classical swine fever (CSF) diagnosis: Technical
Part, 3/2002

37
16. Harding M., 1994. Reverse transcriptase-PCR assay for detection of hog
cholera virus. Jounal of Clinical Biology, 32(10): 2600-2602.
17. Joseph Sambrook v David W. Russell, 2001. Molecular cloning. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
18. Paton D. J, A. McGoldrick, S. Belak, C. Mittelholzer, F. Koenen, H.
Biagetti, G. M. De Mia, T. Stadejek, M. A. Hofmann, B. Thuer, 2000.
Classical swine fever virus: a ring test to evaluate RT-PCR detection
methods. Veterinary Microbiology 73 (2000) 159-174.
19. Paton D. J., McGoldrick, A., Greiser-Wilke, I., Parchariyanon, S., Song,
J., Liou, P. P., Stadejek, T., Lowings, J. P., Bjorklund, H., and Belak, S.,
2000. Genetic typing of classical swine fever virus. Veterinary
Microbiology 73: 137-157.
20. Pereda J. A., 2005. Phylogenetic analysis of classical swine fever virus
(CSFV) field isolates from outbreaks in South and Central America. Virus
Research 110: 111-118.

PH LC
Kt qu phn tch thng k th nghim 1
Kt qu phn tch t s OD
One-Way Analysis of Variance
-------------------------------------------------------------------------------Data: ODM.OD
Level codes: ODM.T
Labels:
Means plot: LSD

Confidence level: 95

Range test: LSD

Analysis of variance
-------------------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio Sig. level
-------------------------------------------------------------------------------Between groups
.9123689
3
.3041230
1.576
.2342
Within groups
3.0882848
16
.1930178
-------------------------------------------------------------------------------Total (corrected)
4.0006538
19
0 missing value(s) have been excluded.
Analysis of Variance for ODM.OD - Type III Sums of Squares
-------------------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio Sig. level
-------------------------------------------------------------------------------MAIN EFFECTS
A:ODM.T
.9123689
3
.3041230
1.576
.2342
RESIDUAL
3.0882848
16
.1930178
-------------------------------------------------------------------------------TOTAL (CORRECTED)
4.0006538
19
-------------------------------------------------------------------------------0 missing values have been excluded.
All F-ratios are based on the residual mean square error.
Table of Least Squares Means for ODM.OD
-------------------------------------------------------------------------------95% Confidence
Level
Count
Average Stnd. Error
for mean
-------------------------------------------------------------------------------GRAND MEAN
20
2.1042500
.0982389
1.8959411
2.3125589
A:ODM.T
1
5
1.8902000
.1964779
1.4735822
2.3068178
1.5
5
1.9896000
.1964779
1.5729822
2.4062178
2
5
2.0822000
.1964779
1.6655822
2.4988178
2.5
5
2.4550000
.1964779
2.0383822
2.8716178

Multiple range analysis for ODM.OD by ODM.T

-------------------------------------------------------------------------------Method: 95 Percent LSD
Level
Count
LS Mean
Homogeneous Groups
-------------------------------------------------------------------------------1
5
1.8902000
X
1.5
5
1.9896000
X
2
5
2.0822000
X
2.5
5
2.4550000
X
-------------------------------------------------------------------------------contrast
difference +/limits
1
- 1.5
-0.09940
0.58919
1
- 2
-0.19200
0.58919
1
- 2.5
-0.56480
0.58919
1.5 - 2
-0.09260
0.58919
1.5 - 2.5
-0.46540
0.58919
2
- 2.5
-0.37280
0.58919
------------------------------------------------------------------------* denotes a statistically significant difference.

Kt qu phn tch hm lng RNA

One-Way Analysis of Variance
-------------------------------------------------------------------------------Data: HLR.RNA4
Level codes: HLR.N
Labels:
Means plot: LSD

Confidence level: 95

Range test: LSD

Analysis of variance
-------------------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio Sig. level
-------------------------------------------------------------------------------Between groups
.7887456
3
.2629152
1.654
.2168
Within groups
2.5438976
16
.1589936
-------------------------------------------------------------------------------Total (corrected)
3.3326432
19
0 missing value(s) have been excluded.
Analysis of Variance for HLR.RNA4 - Type III Sums of Squares
-------------------------------------------------------------------------------Source of variation
Sum of Squares
d.f.
Mean square
F-ratio Sig. level
-------------------------------------------------------------------------------MAIN EFFECTS
A:HLR.N
.7887456
3
.2629152
1.654
.2168
RESIDUAL
2.5438976
16
.1589936
-------------------------------------------------------------------------------TOTAL (CORRECTED)
3.3326432
19
-------------------------------------------------------------------------------0 missing values have been excluded.
All F-ratios are based on the residual mean square error.

Table of Least Squares Means for HLR.RNA4

-------------------------------------------------------------------------------95% Confidence
Level
Count
Average Stnd. Error
for mean
-------------------------------------------------------------------------------GRAND MEAN
20
1.3588000
.0891610
1.1697403
1.5478597
A:HLR.N
1
5
1.4512000
.1783220
1.0730806
1.8293194
1.5
5
1.6144000
.1783220
1.2362806
1.9925194
2
5
1.2928000
.1783220
.9146806
1.6709194
2.5
5
1.0768000
.1783220
.6986806
1.4549194
-------------------------------------------------------------------------------Multiple range analysis for HLR.RNA4 by HLR.N
-------------------------------------------------------------------------------Method: 95 Percent LSD
Level
Count
LS Mean
Homogeneous Groups
-------------------------------------------------------------------------------2.5
5
1.0768000
X
2
5
1.2928000
XX
1
5
1.4512000
XX
1.5
5
1.6144000
X
-------------------------------------------------------------------------------contrast
difference +/limits
1
- 1.5
-0.16320
0.53474
1
- 2
0.15840
0.53474
1
- 2.5
0.37440
0.53474
1.5 - 2
0.32160
0.53474
1.5 - 2.5
0.53760
0.53474 *
2
- 2.5
0.21600
0.53474
-------------------------------------------------------------------------------* denotes a statistically significant difference.

Kt qu phn tch kho st 3

Summary Statistics for Contingency Tables (Page 1)
--------------------------------------------------------------------Chi-square
D.F.
Significance
--------------------------------------------------------------------8.18015
2
0.0167380
WARNING: Expected values in 5 cells < 5 and 1 cells < 2.
With rows
With columns
Statistic
Symmetric
dependent
dependent
--------------------------------------------------------------------Lambda
0.36842
0.33333
0.42857
Uncertainty Coeff.
0.21425
0.17003
0.28957
Somer's D
-0.51613
-0.64286
-0.43114