Trưng ñi hc Nông nghip 1 – Giáo trình Bnh cây ñi cương ---------------------------------------------------
18
MilsteinvKohler(1975)nh−sau:TÕbolymphoBcãgenm?ho¸Ig(t¹okh¸ngthÓ)+
tÕboutuûMyecoma(nh©nnhanh)cñamét®éngvËtbÞungth−.HaitÕbonydunghîp
víinhauv®−îcnu«itrongm«itr−êngHAT(chøahypoxantin,aminorperinvtomidin)
chóngt¹oratÕbolai.ThùchiÖnnu«icÊy®¬nbotrªnb¶nplastictrong®iÒukiÖnv«
trïngtuyÖt®èi tathu®−îc dßng1,2, 3, 4,…Tõ®ãs¶n xuÊt®−îc kh¸ng thÓ®¬ndßng
(monoclonalantibody).Kh¸ngthÓ®¬ndßngcãthÓph¸thiÖntíichñng(strain)cñavirus
haynßi(race)cñavikhuÈnvnÊmg©ybÖnhh¹ithùcvËt.
Kh¸ngnguyªn(viruscãtrongdÞchl¸bÖnh)sÏkÕthîpvíikh¸ngthÓ(cãtrongkh¸ng
huyÕtthanh)®Òut¹okÕttñadïlkh¸ngthÓ®¬ndßnghay®adßng.
Kh¸nghuyÕtthanhlu«ncãtÝnh®ÆchiÖucao:
ekh¸ngnguyªnAchØkÕthîpvíikh¸ngthÓA.
ekh¸ngnguyªnBchØkÕthîpvíikh¸ngthÓB.
ekh¸ngnguyªnCchØkÕthîpvíikh¸ngthÓC.
Kh«ngcãhiÖnt−îngkÕttñachÐogi÷aA,B,C,chÝnhv×vËychóngtacãthÓsödông
kh¸nghuyÕtthanh®ÓchÈn®o¸nx¸c®ÞnhbÖnhh¹i.Trongsuètnh÷ngn¨m®ÇucñathÕkû
20(1930e1970)kh¸nghuyÕtthanhlph−¬ngph¸pchÈn®o¸nrÊtquanträngv×ph¶nøng
x¶yranhanhtõ15e20phóttrong®iÒukiÖnnhiÖt®éphßngthÝnghiÖmkho¶ng20e25
0
C.
Nh−ng®Óquans¸tph¶nøngngymétkhãh¬nkhitagÆpc¸ctr−ênghîpkÕttñaqu¸Ýt
khãcãthÓph¸n®o¸ncãhaykh«ngcãph¶nøng(ph¶nøng±).N¨m1977,ClarkvAdam
(Scottlen) ®? dïng ph−¬ng ph¸p thö nghiÖm miÔn dÞch liªn kÕt men (Enzyme linked
immunosorbentassay–ELISA)lÇn®ÇutiªntrªnthùcvËt.Ph−¬ngph¸pELISA®?t¹ora
mét sù ®æi míitrong viÖc södông kh¸ng huyÕt thanh lm t¨ng®é chÝnh x¸c lªnhng
ngh×nlÇn.B¶nchÊtcñaph−¬ngph¸pvÉnlsödôngkh¸ngnguyªnvkh¸ngthÓt¹orasù
kÕthîpgi÷achóngvíimen(enzyme)liªnkÕtenh−ngchØthÞcñaph¶nøngkh«ngph¶il
kÕttñamlmuvng.M¸y®äcELISA®?kh¾cphôchiÖnt−îngmuvngnh¹tdÇnv
cungcÊpchochóngta b¶ngsèliÖu chØrâ c¸cph¶nøngx¶yraëtõnggiÕng trongb¶n
ELISA.
Ph−¬ngph¸pELISAdirect(DASeELISA),ph−¬ngph¸pindirectlnh÷ngph−¬ng
ph¸psödôngphæbiÕntrªnthÕgiíicho®Õnnay,nh÷ngph−¬ngph¸pnyvÉndïngréng
r?itrongs¶nxuÊtv®−îccoilph−¬ngph¸phuyÕtthanhchÝnhx¸cnhÊt®−îcsödông
hiÖnnay.ChitiÕtc¸cquytr×nhcñaph−¬ngph¸pELISAnh−sau:
Ph−¬ngph¸pDASHELISA(DoubleantibodysandwichHELISA)haycßngäi
l)ph−¬ngph¸pELISAtrùctiÕp
B−íc1:Cè®ÞnhIgG®ÆchiÖucñavirusvob¶nELISA.
IgGhotrongdungdÞch®Ömcarbonate,chovomçigiÕng100µl.§Ætb¶nELISA
tronghépÈm,®ÓvotñÊmënhiÖt®é37
0
Ctrongthêigian2e4h.Saukhiñ,giÕng®−îc
röab»ngdungdÞch®Ömröa(PBSeT)balÇn,mçilÇntrong3phót.
Trưng ñi hc Nông nghip 1 – Giáo trình Bnh cây ñi cương ---------------------------------------------------
19
B−íc2:Cè®ÞnhdÞchc©yvob¶nELISA.
NghiÒnmçimÉu2gtrong®ÖmchiÕt(PBSeT+2%PVP)víi®éphalo?ng1/10v
1/20.DÞchc©y®−îcnhávob¶nELISAvíil−îng100µl/giÕng.Sau®ã®Ætb¶nELISA
vohépÈm®Ótrongtñl¹nhë–4
0
Cquamét®ªmhoÆccãthÓñë37
0
Ctrongkho¶ng2e
4h.Trongqu¸tr×nhnyIgGsÏx¶yraliªnkÕtgi÷aIgGvkh¸ngnguyªn(nÕumÉulmÉu
bÞnhiÔmbÖnh).Saukhiñb¶nELISA®−îcröanh−b−íc1.
B−íc3:Cè®ÞnhIgGliªnkÕtenzyme.
HoIgGliªnkÕtenzyme(IgGeE)trongdungdÞch®ÖmliªnkÕt(PBSeT+2%PVP
+0,2%Ovalbumin)theotûlÖchovogiÕngvíil−îng100µl/giÕng.B¶nELISA®−îcñë
37
0
Ctrong2hvröanh−b−íc1.
B−íc4:Cè®ÞnhchÊtnÒnvob¶nELISA.
HochÊtnÒnNPP(nitrophenolphosphate)vodungdÞch®Ömsubstrate(theotûlÖ
0,25e0,5mg/1mldungdÞch®Öm).Sau®ãnhávomçigiÕng100µl.B¶nELISA®Ótrong
hépÈm ®−îc®Æt ënhiÖt ®étrong phßng thÝ nghiÖm.Sau1hc¸c giÕngcã mu vng l
giÕngcãph¶nøngd−¬ngtÝnh,giÕngkh«ngmulkh«ngcãph¶nøng.KÕtqu¶®−îc®äc
chÝnhx¸ch¬ntrªnm¸y®äcELISA(ELISAreader)ëb−ícsãng405nm.
§Ócè®Þnhmus¾ccñab¶nELISA,b¶oqu¶ntrongtñl¹nh4
0
CnÕucÇnxeml¹ivo
khikh¸ccãthÓdïngdungdÞchNaOH3MnhávomçigiÕng25e30µl.
Ph−¬ngph¸pIndirectELISAhaycßngäil)ph−¬ngph¸pELISAgi¸ntiÕp
B−íc1:Cè®ÞnhdÞchc©y(nghilbÞbÖnh)cÇnkiÓmtravob¶n:cÇnmçimÉu0,2g
l¸chovotóinilonnghiÒntrongdungdÞchPBSvíitûlÖl¸/dungdÞch®Öml1/20e1/100,
nhávob¶n100µl/giÕng.Sau®ã®Ób¶nELISAvohépÈmvñquamét®ªmënhiÖt®é
4
0
C.
B−íc 2: ChuÈn bÞ mÉu c©y khoÎ (c©y ®? ®−îc kiÓm tra ELISA kh«ng bÞ nhiÔm)
nghiÒn trong dung dÞch ®Öm pha huyÕt thanh (PBS e T 1000ml + 2% PVP + 0,2%
Ovabumin)theotûlÖ1/20.Läcquav¶iläctathu®−îcdÞchc©ykhoÎ.Chokh¸nghuyÕt
thanhvodÞchc©ykhoÎtheonång®é®?phalo?ngtuútõnglo¹ikh¸nghuyÕtthanhkhuÊy
®Òuv®Ó45phóttrong®iÒukiÖn37
0
C.
B−íc3:Röab¶nELISAvíi®ÖmPBSeT3lÇntrong3phót.
B−íc4:Cè®Þnhkh¸nghuyÕtthanhvob¶nELISA,nhávomçigiÕng100µlkh¸ng
huyÕtthanh®?phalo?ngtrongdÞchc©ykhoÎ.Sau®ãchob¶nELISAvotronghépÈmv
®Óë®iÒukiÖnnhiÖt®é37
0
Ctrongthêigiantõ1e1h30phót.
B−íc5:Röab¶nELISAnh−ëb−íc3.
B−íc 6: Cè ®Þnh kh¸ng huyÕt thanh cña kh¸ng nguyªn IgG thá (hay kh¸ng huyÕt
thanh B (conjugate AP) víi ®é ho lo?ng 1/1000 e 1/2000. Mçi giÕng 100 µl. §Æt b¶n
Trưng ñi hc Nông nghip 1 – Giáo trình Bnh cây ñi cương ---------------------------------------------------
20
ELISAvohépv®Óquamét®ªmëtñl¹nh4
0
C(hoÆc®ÓënhiÖt®é37
0
Ctrong1he1h30
phót).
B−íc7:Röab¶nELISAnh−b−íc3.
B−íc8:Cè®ÞnhchÊtnÒnv®¸nhgi¸kÕtqu¶:
ePha0,25e0,3mgNNP/1ml®Ömsubtrateråihotanb»ngm¸ykhuÊytõ.
eSau®ãnhádungdÞchtrªnvob¶nELISA,100µl/giÕng.
§−ab¶nELISAvohépÈmv®ÓtrongnhiÖt®éphßngthÝnghiÖm(kho¶ng20
0
C)
trongthêigiantõ30e60phót.
B−íc9:§äckÕtqu¶:
C¸cgiÕngcãmuvnglc¸cgiÕngcãph¶nøng(+).GiÕngkh«ngcãmulc©y
kh«ngbÞnhiÔmbÖnh.§äckÕtqu¶tiÕpb»ngc¸ch®−avom¸y®äcELISAëb−ícsãng
405 nm. Còng cã thÓ dõng ph¶n øng b»ng NaOH 3M víi l−îng 25 e 50 µl/giÕng nh−
ph−¬ngph¸pDASeELISA.
e.C¸cph−¬ngph¸pchÈn®o¸nsinhhäcph©ntö
Ph−¬ngph¸pchÈn®o¸nkh¸nghuyÕtthanhvELISAlnh÷ngph−¬ngph¸pchÈn
®o¸nprotein.Chotíinay(2006)vÉnlph−¬ngph¸p®−îcøngdôngréngr?i®ÓchÈn®o¸n
virusëng−êi,®éngvËtvthùcvËt®?®−îngc¸ch?ngAgdia,Biorad(Mü),nhiÒuh?ngs¶n
xuÊt cña NhËt, §øc, Ph¸p, H Lan...th−¬ng m¹i ho¸ v®−a ra thÞ tr−êng rÊt nhiÒu s¶n
phÈmdogi¸trÞcñac¸cs¶nphÈmnyrÎv®échÝnhx¸ccao.Nh÷ngph−¬ngph¸pchÈn
®o¸n sinh häc ph©n tö l ph−¬ng ph¸p ph¸t hiÖn ARNv ADN. Tõ nh÷ng n¨m 80 khi
ph−¬ngph¸psinhhäcph©ntöra®êith×viÖcx¸c®ÞnhvirusthùcvËtb¾t®Çuph¸ttriÓnë
møc®éph©ntö.CãrÊtnhiÒuph−¬ngph¸psinhhäcph©ntö®−îcøngdông,songtíinay
PCR(polymerazachainreaction)lph−¬ngph¸p®−îcsödôngréngr?ivmangl¹ihiÖu
qu¶caonhÊt.Ph−¬ngph¸p®−îcthùchiÖntrªnc¬sëkh¶n¨ngt¸itæhîpcñaADN,ARN
invitro.MuènthùchiÖnkh¶n¨ngnycÇncãc¸c®iÒukiÖnc¬b¶nsau:T¸ch®−îc1l−îng
nháADNnguyªnb¶n,trénvíiméttËphîpc¸cchÊttrongm«itr−êngmuèi®ÖmgåmTaq
Polymeraza,dNTPs(Deoxyribonucletittriphophates),MgCl
2
.MéthoÆchai®o¹nnucleotit
lmmåi(primer).
Tãmt¾tc¸cb−íccñaph−¬ngph¸pPCRgåm:
eB−íc1:SîiADNkÐp®−îcxölýë94
0
Ctrong5phótt¹othnh2sîi®¬n.
eB−íc2:§o¹nbæ sungsîi®¬n ADNv®o¹n måighÐpcÆpë30 e65
0
Ctrong3
gi©y.
eB−íc3:Tænghîpsîi®¬nmíiADNë65–75
0
Ctrong2e5phót.
eB−íc4:Quaytrël¹ib−íc2saukhiADNkÐpl¹it¸chthnh2sîi®¬në94
0
Ctrong
30gi©y….s¶nphÈmPCR®−îc®iÖnditrªngelAgarosehoÆcgelPolyacrylamide.
Trưng ñi hc Nông nghip 1 – Giáo trình Bnh cây ñi cương ---------------------------------------------------
21
g.Ph−¬ngph¸phiÓnvi®iÖntö
Ph−¬ngph¸pkÝnhhiÓnvi®iÖntölph−¬ngph¸pquanträng®Óph¸thiÖnc¸cvirus,
phytoplasma,viroideg©ybÖnhëthùcvËtmkÝnhhiÓnvith«ngth−êngvíi®éphãng®¹i
nhákh«ngthùchiÖn®−îc.
MaxKnol,ErnettRuska(1931)lnhãmc¸cnhkhoahäc®?chÕt¹othnhc«ng
kÝnhhiÓnvi®iÖntö.Tõ®ã,virusthùcvËtv®éngvËt®?®−îcquans¸tchÝnhx¸cvÒh×nh
th¸i,cÊut¹o.HiÓnvi®iÖntöcãhailo¹ikÝnhchñyÕul:
KÝnhhiÓnvi®iÖntötruyÒnqua(transmissionelectronmicroscope)vhiÓnvi®iÓntö
quÐt(scaningelectronmicroscope).KÝnhhiÓnvi®iÖntötruyÒnquallo¹ikÝnh®−îcsö
dôngphæbiÕntrongnghiªncøubÖnhc©y.§ÓthùchiÖn®−îcküthuËtquans¸tvchôp
¶nhhiÓnvi®iÖntöcãthÓxemtrùctiÕpmÉu(ph−¬ngph¸pDIP)hoÆcxemmÉuvisinhvËt
(chñyÕulvirus)®?®−îclmtinhkhiÕt.HoÆcc¾tl¸tc¾tm«bÖnhcùcmángb»ngm¸y
c¾tsiªumáng(Ultramicrotom)®Óquans¸tvirustrongm«tÕboc©ybÞnhiÔmbÖnh.TÊt
c¶c¸cph−¬ngph¸pt¹omÉutrªn®ÒusödôngnhiÒuthuècnhuémvho¸chÊt®Ócè®Þnh
mÉuvËtvkhithùchiÖnl¸tc¾tsiªumángph¶itiÕnhnhvíim¸yc¾ttrongch©nkh«ng.
Tõph−¬ngph¸phiÓnvi®iÖntölmc¬sëngynay®?ra®êivph¸ttriÓnnhiÒuküthuËt
míinh−hiÓnvilùcnguyªntö,hiÓnvitõlùc,hiÓnviquÐthiÖuøng®−êngngÇm.
h.C¸cph−¬ngph¸pkh¸c
§èivíimétsèbÖnhh¹ithùcvËttr−íc®©yng−êita®?dïngmétsèph−¬ngph¸p
®¬ngi¶nvíi®échÝnhx¸ckho¶ng80%®ÓchÈn®o¸ns¬bébÖnhh¹i:
−
DungdÞchRezocin10%khinhuémmul¸tc¾tmángëcñkhoait©yph¸thiÖnthÊy
c¸cbãm¹chlibebÞnhuémmuxÉmlhiÖnt−îngcñ®?bÞnhiÔmviruscuènl¸(Potato
leafrollvirusePLRV).
−
DungdÞchsunfat®ångCuSO
4
.5H
2
O3%nhuémmun©u®ákhixölým«c©yhä
c,häbÇubÝcãthÓph¸thiÖnsùnhiÔmbÖnhdovirusCucumbermosaic(CMV).
−
Ph−¬ng ph¸p gi¸m®Þnh nhanh bÖnh vng l¸ Greening b»ngnhuém Iod v dïng
giÊythöNCMchokÕtqu¶tèt.
−
Ph−¬ngph¸p®o®énhít,®é®ôccñadÞchc©ycònglph−¬ngph¸pchÈn®o¸ns¬
bébÖnhh¹itrongtr−ênghîpc©ybÞbÖnhdÞchc©yth−êngcã®é®ôccaoh¬n.
−
Ph−¬ngph¸phuúnhquang®ÓchÈn®o¸nm«qu¶,h¹tbÞbÖnhdùavosùph¸ts¸ng
cñam«bÖnhkhitachiÕmnguåns¸ngtõ®Ìnth¹chanhcãb−ícsãngkh¸cnhau....
−
Ph−¬ngph¸pchÈn®o¸nbÖnhtrªnh¹tgièngtr−íckhigieotrång:SaukhilÊymÉu
kiÓmtrah¹ttheoquy®Þnh,cãthÓthùchiÖnph−¬ngph¸pröah¹tråilyt©mnhÑvquan
s¸tdÞchthu®−îctrªnkÝnhhiÓnvi®Óph¸thiÖnbotöhaysîinÊmbÖnhh¹i.Dïngc¸ch
khötrïng®ÊtthÝnghiÖmråing©mñh¹tvgieotrªn®Êtv«trïngph¸thiÖnc©yconbÞ
bÖnh.Dïngph−¬ngph¸pELISAvPCRchÈn®o¸nbÖnhëcñhayh¹tgièng.
Trưng ñi hc Nông nghip 1 – Giáo trình Bnh cây ñi cương ---------------------------------------------------
22
−
VíituyÕntrïng:ng−êitalÊymÉu®ÊtëchiÒus©utõ5–20cmvsödôngph−¬ng
ph¸pBekman(1995).LäctuyÕntrïngqual−íiläc25µmsaukhi®?ng©mmÉu®Êttõ24e
48h.HoÆcng©mrÔc©ytrongcècn−íc2e3htathu®−îctuyÕntrïngë®¸ycècvdïng
kÝnhlópphãng®¹i50lÇn®Óquans¸t.
−
Ph−¬ngph¸pph¸thiÖngiätdÞchvikhuÈn:cãthÓdïngl¸(bÖnhb¹cl¸lóa)hayth©n
(bÖnhhÐoxanhc©yhäc)ng©mvodungdÞch1%NaClhoÆcn−ícs¹ch,sau20e30
phótsÏthÊygiätdÞchvikhuÈnxuÊthiÖnë®Çul¸hayël¸tc¾tth©nnh«lªnmÆtn−íc.