NGHN CU CH TO DNA-MCROARRAY PHT HN V CHN ON

NHANH CC TEM BETA-LACTAMASE KHNG KHNG SNH PH RONG NHM

BETA-LACTAM
Bi Vn Ngc
1
, Nghim Ngc Minh
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, Nguyn Hoi Thu
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, Nguyn Hu c
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, Nguyn c Thng
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, Nguyn !ng
Hi"u
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Vin Cng ngh sinh hc - Vin HL KH v CNVN
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Khoa Cng ngh sinh hc - i hc Nng nghip H Ni
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Khoa Cng ngh Thc ph v H!a hc - i hc "ao #
TM TT
Tnh khng khng sinh nhm beta-lactam nhiu loi vi khun l do chng sn sinh cc beta-lactamase ph !ng c nhi"m v# th$%
ph&n' ph v( c)u tc c$a khng sinh* +!t tong cc beta-lactamase , l T-+* .o ,!t bi/n 0% a m!t s1 codon lm tha% ,i
amino acid tong tung t&m ho2t ,!ng c$a beta lactamase d3n ,/n h4nh thnh nhiu bi/n th5 T-+ khc nhau khng l2i ph khng
sinh !ng h6n* Tong nghi7n c8u n%' .9:-micoaa% ,;<c ch/ t2o ,5 pht hi"n nhanh v chn ,on hai bi/n th5 c$a T-+ l T-+-
==> v T-+-? th@ng Aua vi"c 0c ,Bnh tnh ,a h4nh nucleotide ,6n CD9EF t2i nGm vB t t7n t4nh tH ,ch blaT-+-==> v blaT-+-?*
.Ha vo Au t4nh bIt cJp b sung hon ton v kh@ng hon ton giKa cc m3u dL t7n micoaa% vMi t4nh tH ,ch ,N 0c ,Bnh ,;<c
cc vB t D9E v k/t luOn nucleotide bB tha% th/ C,!t bi/nF ha% bo tPn* T7n blaT-+-==>' cc base Q v R t2i vB t D9E ?S v =?S
,;<c tha% th/ bTng : v T t2o thnh hai codon mMi' d3n ,/n tha% th/ amino acid t2i hai vB t t;6ng 8ngU ValC?SFWle v :laC=?SFVal*
Tong khi ,' t7n blaT-+-? t2i vB t D9E =>S*= v SX>' R v Q ,;<c tha% th/ bTng : v :' do vO% cYng h4nh thnh hai codon mMi
t;6ng 8ng vMi sH tha% th/ amino acidU :gC=>SFDe v Ql%CSX>FDe*
T kha: .9:-+icoaa%' beta-lactamase ph !ng' blaT-+' ,a h4nh nucleotide ,6n' bIt cJp b sung hon ton v kh@ng hon
ton' c%anine
M# $%
Mt trong nhng mi nguy him ln nht trong qu trnh chm sc sc khe cng dng l tnh khng khng sinh nhm
beta-lactam ph rng ca mt s vi khun gy bnh thuc ho $n%&'o(ac%&'iaca& nhu $sch&'ichia co)i , K)&(si&))a
pn&*onia& , +s&*,oonas a&'*ginosa . Kh nng khng khng sinh ca cc loi ny l do chng sn sinh ra cc
enzym beta-lactamase ph rng (Extended-spectrum beta-lactamase - ESBL) c kh nng thy phn vng -lactam v
ph v cu trc ca khng sinh . Cc beta-lactamase din hnh duoc bit dn nhu TEM (Temoniera), SHV (sulphydryl
variable), CTX-M (CefoTaXime-Munich) duoc m ha bi cc gen tuong ng l ()aTEM, ()aSHV, ()aCTX-M. Do duoc truyn
qua plasmid nn cc beta-lactamase ny duoc tm thy trong nhiu loi $n%&'o(ac%&'iaca& nhiu quc gia trn th gii
. Thm vo d, do dt bin lm thay di nucleotide trong mt s codon m ha cho amino acid thuc trung tm hoat
dng ca enzym, dn dn thay di thnh phn amino acid v tao ra cc bin th enzym khc nhau c kh nng ph hy
ph khng sinh nhm beta-lactam rng hon, khng chi ampicillin, penicillin, m cn bao gm cc oxyimino-
cephalosporin (ceftazidime, ceftriaxone, cefepime, cefpodoxime, cefotaxime.). thm ch carbapenem. Cho dn nay,
c khong 140 bin th ca TEM-1, 60 bin th ca SHV-1 v 80 bin th ca CTX-M-1 duoc tm thy. Thm vo d,
su tuong dng v trnh tu amino acid gia TEM-1 v SHV-1, gia TEM-1 v CTX-M ln luot l 68%, 40% . V vy, vic
chn don tnh khng beta-lactam mt s loi vi khun gy bnh sinh beta-lactamase ph rng ni chung v cc bin
th ca TEM ni ring l mt trong nhng chin luoc quan trong trong diu trj v phng chng bnh tt.
Truc dy, trong pht hin v chn don ngui ta c th dng phuong php vi sinh hoc lm sng, nui cy v quan st
hnh thi dui knh hin vi . Phuong php ny tuy don gin, nhung cn t nht 2 ngy v c th khng phn bit duoc
mt s loi dua vo dc dim hnh thi. Bn canh d, phuong php Vitek ESBL cng duoc dng d pht hin mt s
loi vi khun sn sinh nhm beta-lactamase , tuy nhin phuong php ny c d nhay thp. Gn dy, cc phuong php
PCR duoc pht trin nhu PCR-RFLP , PCR-SSCP hoc RT-PCR . tuy rt ngn thi gian pht hin, cng nhu tng d
nhay v dc hiu dng k so vi cc phuong php trn, nhung khng linh dng trong vic phn tch song song, cng
mt lc cc bin th nhiu chng loi hay tnh da hnh nucleotide don (single nucleotide polymorphism, SNPs). Trong
khi d, DNA-Microarray c kh nng pht hin nhanh trong khong 4 h, dc bit chn don dng thi cc bin th
enzym v tnh da khng thuc ca nhiu loi trong cng mt lc . iu d gip cho vic diu trj kjp thi v chnh xc v
trnh lp lai th nghim nhiu ln. Chnh v th, trong nghin cu ny, chng ti buc du nghin cu ch tao DNA-
Microarray d pht hin v chn don nhanh hai bin th ca TEM-1 l TEM-116 v TEM-8, duoc tch ra t $- co)i v
K- pn&*onia&.
N&%'(N )*+% V, -H./N& -H0-
Nguyn 1i2u
Tt c cc ha cht s dung trong nghin cu ny du duoc mua t hng Sigma-Aldrich, Merck, BASF, nvitrogen, New
England Biolabs (c). Cc gen ()aTEM-1 (014), ()aTEM-116 (U611) v ()aTEM-8 (X622) duoc tch ra t hai
loi vi khun gy bnh $- co)i v K- pn&*onia&, sau d duoc gn ln plasmid pUC1 (New England Biolabs) v bin
nap vo $- co)i DH (Clontech), cng nhu cp mi dng d khuch dai cc gen ny du sn c tai nhm nghin cu
ca TS. Till T. Bachmann thuc Vin Ha sinh k thut (TB), H Stuttgart, c (hin nay thuc H Edinburgh - Anh).
-h34ng 5h65
1
Thit k mu d pht hin tnh a hnh nucleotide n (SNPs
Trnh tu cc gen ()aTEM-1 (014), ()aTEM-116 (U611) v ()aTEM-8 (X622) d cng b trn ngn hng gen
(NCB) duoc so snh bng cng cu sp thng hng da trnh tu (phn mm BioEdit hoc CLC Sequence Viewer) d xc
djnh su khc bit nucleotide hay tnh da hnh nucleotide don (SNPs) lm thay di amino acid trong trung tm hoat dng
ca hai bin th ny (Bng 1). Nucleotide khc bit no dn dn su thay di amino acid s duoc dt vo vj tr gia ca
mu d, trong d mu d l doan oligonucleotide c kch thuc t 1-1 base. Do vy, d xc djnh mt SNP, cn s
dung 4 mu d ging nhau v trnh tu, nhung khc nhau nucleotide dt vj tr gia mu d.
B7ng 18 T9:nh ;< m=u >? @3Ac >ng @B 5h6; hi2n CN- ;9ong c6c Di"n ;hB cEF TGM Hh6c nhFu
Vj tr codon trn ()aTEM-1 m
ha amino acide
Trnh tu mu d (-)
82 (GTT) TTATCCCGTNTTGACGCCG
162.1 (CGT) GCCTTGATN&TTGGGAA
162.2 (CGT) GCCTTGATNTTGGGAA
182 (GCA) CGATGCCT&NIGCAATGGC
26 (GGT) CTGGAGCCN&TGAGCGT
() Nucleotide bin di trong b ba m ha lm thay di amino acid duoc gach chn
() Mi trnh tu mu d s tao thnh 4 mu d khc nhau, chng ging nhau trnh tu nucleotide, nhung khc nhau vj tr N (A, T, G
hoc C), tc tng s mu d s l 20 trnh tu.
T!o "N#$%ic&oa&&a'
Cc mu d sau khi thit k v dt mua (nvitrogen, c) duoc s dung d pht hin cc SNP (Bng 1) s duoc gn bng
cch dp (printing) hay chm (spotting) ln b mt ca phin knh (glass silde) c kch thuc 2x22x1 mm (Nexterion Slide
E, Schott, ena, c) bng robot c 8 ghim (BioRobotics MicroGrid , Genomic Solutions Ltd., Huntingdon, Anh). Mi chm
s duoc dp ln. Sau khi dp mu d ln b bt, ton b phin knh duoc tai 60C 0 min. Sau d duoc ra ln, ln
1 trong nuc ct hai ln b sung 0,1% Triton X-100 min, ln 2 trong nuc ct hai ln b sung 0,0% HCl dm dc (%)
4min, ln trong dung djch mui KCl 100 mM 10 min. Tip theo cc phin knh ny duoc ngm tip trong dung djch
cha 2% ethylen glycol v 0,0% HCl dm dc tai 0 C trong 1 min. Cc phin knh tip tuc duoc ra bng nuc ct
hai ln trong 1 min, sau d duoc lm kh bng kh nito v c th s dung ngay hoc bo qun trong diu kin ti vi han
s dung ti da 20 ngy. u trnh chm mu d ln b mt ca phin knh duoc m ta theo hnh 1a v DNA-Microarray
duoc tao ra d pht hin mt SNP duoc m t nhu hnh 1b. Ton b qu trnh ny duoc lp lai hai ln, do vy mi dim
trn microarray s c 6 gi trj khi do d ly gi trj trung bnh (Hnh 2b).
(hu)n *+, tinh s!ch -. /0 l1 t&nh t2 ch
Cc trnh tu dch (phn nguyn liu) duoc khuch dai v dnh du bng cht nhum cyanide (Cy) thng qua PCR, bao
gm cc thnh phn 2 ng plasmid 0,4 M mi mi loai dm 1xPCR cha 2, mM Mg(C2HO2)2, 0 mM KCl, 10
mM Tris-HCl pH 8, 0 M dNTPs (dATP, dGTP, dTTP) 0 M dCTP 20 M Cy-dCTP 1 U Taq DNA polymerase.
Phn ng duoc thuc hin tai 4 C - 1 min 0 chu k lp lai (4 C - 0 s, C - 0 s, 2 C - 1 min) 2 C - 4 min
(Mastercycler, Eppendorf, c). Sn phm PCR (kch thuc 86 bp) sau khi dnh du v tinh sach (iaquick Spin PCR
purification kit, iagen) s duoc pha long ti nng d 0 ngl trong dung djch dm (40 mM Tris-HCl pH 8, 10 mM
MgSO4, 1 mM CaCl2), sau d duoc x l bng enzym DNase (11, mUl, Promega, Mannheim, c) d phn ct
thnh cc doan oligonucleotide dch c kch thuc khc nhau phuc vu cho qu trnh lai sau ny. Cui cng, b sung
mM EGTA v tai 6 C trong 10 pht d bt hoat enzym DNase .
3ai ph4n t0
2100 ng cc doan oligonucleotide dch cng vi 0,0 pmol trnh tu bt cp trong lai di chng duong (Hnh 1a, pos.
Hybridisierungs-Kontrole) s duoc lai vi cc trnh tu trn mi phin knh (microarray, Hnh 1b) trong 6 l dung djch
2xSSPE (0,6 M NaCl 20 mM NaH2PO4 2 mM EDTA pH ,) c b sung thm 0,1% SDS. u trnh lai duoc thuc hin
trong t lai n nhit (OV, Biometra) tai 4 C trong 2 h. Khi qu trnh lai kt thc, cc phin knh s duoc ra hai ln
nhit d phng ln 1 trong 2xSSC (0, M NaCl v 0,0 M Natri citrate) b sung 0,1% SDS trong 1 min ln 2 trong
0,2xSSC trong 1, min. Sau d, cc phin knh ny duoc lm kh bng kh nito trong 4 min v duoc dem di xc djnh tn
hiu hunh quang ca Cy. u trnh lai duoc minh hoa trn hnh 2a.
5o c67n8 9 hu:nh ;uan8, tnh ton, /0 l1 -. *iu di<n s= liu
Tn hiu hunh quang pht ra ca Cy duoc do tai hai buc sng 0 nm (sng kch thch) v 0 nm (sng pht xa)
bng my ScanArray Express Microarray Scanner (PerkinElmer, altham, MA). Kt qu duoc ghi lai dui dang nh (pg
hoc png, hnh 2b) bng phn mm ScanArray Express, dng thi tn hiu cung d hunh quang duoc chuyn v truy
xut dui dang bng tnh Microsoft Excel. Cung d tn hiu hunh quang thuc tai mi dim duoc tnh bng cch tr di
cung d tn hiu hunh quang ca mi trung xung quanh (background). Cung d tn hiu thuc ca mt trong bn vj
tr nucleotide (A, G, C hoc T) tai mi vj tr SNP cao nht s tuong ng vi bt cp hon ton tai vj tr nucleotide d
(perfect match PM, hnh 2a) v duoc goi l PM, tai ba vj tr nucleotide cn lai c cung d thp hon tuong ng vi cc
cp b sung khng hon ton (mismatch MM, hnh 2a) v duoc goi l MM. V th, PM s lun c gi trj ln hon MM.
thun loi cho vic xc djnh PM hay MM tai mi vj tr SNP, cung d tn hiu ca bn nucleotide tai mi vj tr SNP duoc
chun ha thng qua t s PMPM v MMPM. Do d, t s PMPM 1 cng tuong ng vi bt cp b sung hon ton v tai
duy nht mt trong bn nucleotide (A, G, C hoc T), trong khi MMPM 1 tuong ng vi bt cp khng hon ton tai ba vj
tr nucleotide cn lai. Kt qu cui cng duoc biu din trn biu d Microsoft Excel sau khi ly gi trj trung bnh tai 6
dim do cho mi vj trj SNP v d lch chun.
2
H:nh 18 6ch DJ ;9K LM ;9K c6c m=u >? NFO PQ @3Ac >R5 ;9n 5hi"n HKnh @B ;So ;hnh mic9oF99Fy NDO8 Mi vj tr SNP cn pht hin s
duoc dp lp lai ln v duoc ghi trn mi vj tr SNP tuong ng (a), cc base A, G, C, T nm ct tri ngoi cng ca (a) s nm tuong
ng vj tr gia ca trnh tu mu d trn (b), (b) minh hoa microarray pht hin SNP tai codon 26 ( GGT), trong d base cn kim tra
duoc gach chn. Druck-Kontrolle l vj tr trnh tu d duoc gn cht nhum hunh quang d dm bo hoat dng ca qu trnh dp bng
robot neg. Hybridisierungs-Kontrole noi trnh tu khng bt cp vi trnh tu no trong qu trnh lai pos. Hybridisierungs-Kontrole noi
trnh tu chc chn bt cp vi trnh tu cho truc d gn cht nhum hunh quang trong qu trnh lai prozess-Kontrolle noi trnh tu bt
cp vi trnh tu bo th ca ()aTEM trong qu trnh lai. Cc trnh tu nhu d m t trn (a) duoc s dung d kim tra v dm bo d tin
cy ca qu trnh chm mu d ln phin knh, qu trnh lai bt cp vi cc trnh tu d chun bj truc v khng lin quan dn trnh tu
dch cn tm.
H:nh 2T Minh hF Uu6 ;9:nh 1Fi 5hVn ;W ;Si CN- 23X ;9n mic9oF9Fy NFO L H"; Uu7 ;hu @3Ac PFu Hhi @o ;Kn hi2u huYnh UuFng NDO8
u trnh bt cp, b sung hon ton v khng hon ton gia cc trnh tu ca mu d trn microaray vi cc doan duoc dnh du
bng Cy ca trnh tu dch cn tm (a). Sau khi lai v ra, microarray duoc quyt bng chm sng trong my do d ghi lai cung d
hunh quang pht ra tai mi dim (b). Mi dim do trn hnh 2b tuong ng vi cch b tr v thit k trn hnh 1a.
Z[T \%] V, TH]^ )%_N
Cc bin th ca TEM-beta lactamase duoc pht hin thng qua vic xc djnh tnh da hnh nucleotide don (SNP) tai
mt s vj tr trn trnh tu dch ()aTEM dua vo qu trnh lai phn t v bt cp b sung gia cc mu d trn microarray
vi cc trnh tu dch duoc dnh du bng cht nhum pht hunh quang. Sau khi ra chon loc trong dung djch mui c
nng d gim dn s loai b cc doan khng bt cp b sung, m cn lai ch yu cc cp d b sung hon ton (c
cung d tn hiu cao nht, PM) v b sung khng hon ton (c cung d tn hiu thp hon, MM). Dua vo vic so snh
cung d tn hiu gia cc dim thng qua t s PMPM v MMPM s xc djnh duoc vj tr SNP v kt lun nucleotide bj
thay th (dt bin) hay gi nguyn (bo tn)
T kt qu tnh ton t s PMPM v MMPM kt hop so snh vi vj tr SNP cn xc djnh trn ()aTEM-1 l 82(GTT),
162.1(CGT), 162.2(CGT), 182(GCA) v 26(GGT) cho thy
i vi ()aTEM-116 (hnh , pha trn) tai vj tr SNP 82, base A c cung d tn hiu cao nht so vi cc base khc l
G, C, T. iu d c ngha qu trnh bt cp b sung gia mu d v trnh tu dch l hon ton tai vj tr base A (nm vj
tr gia trnh tu mu d), trong khi d qu trnh bt cp b sung khng hon ton xy ra tai cc vj tr G, C, T. V vy, tai
vj tr SNP 82 ca ()aTEM-116 xut hin base mi l A. Ni khc di G trn ()aTEM-1 duoc thay bng A trn ()aTEM-116,
dn dn hnh thnh codon mi l ATT trn ()aTEM-116 v duoc vit thnh GTT(82)ATT. Trong khi d, tai SNP 162.1,
base C c cung d tn hiu cao nht tuong ng vi qu trnh lai hon ton tai C v qu trnh lai khng hon ton tai A,
G, T. Do vy, trn ()aTEM-116 khng xut hin base mi tai SNP 162.1 hay codon 162 ca ()aTEM-116 vn duoc bo
tn l CGT. Ging nhu SNP 162.1, SNP 162.2(CGT) trn ()aTEM-116 vn duoc bo tn. Tuy nhin, tai SNP 182 trn
()aTEM-116 xut hin base mi l T. Do d, base C trn ()aTEM-1 duoc thay th bng base T trn ()aTEM-116, tao
thnh codon mi l GTA v duoc vit l GCA(182)GTA. Tri lai, khng c su thay th base no xy ra tai SNP 26
(hnh , trn). Nhu vy, so snh vi cc vj tr SNP trn ()aTEM-1, trn ()aTEM-116 xut hin hai base mi l A v T tai
hai vj tr SNP 82 v 182 tao thnh hai codon mi tuong ng vi hai vj tr thay th l GTT(82)ATT v GCA(182)GTA. Su
thay th ny dn ti su thay di thnh phn amino acid tuong ng l Val(82)le v Ala(182)Val v qua d tao thnh bin

th beta-lactamase mi vi tn goi TEM-116 .

Theo cch lp lun tuong tu nhu trn v so snh vi cc vj tr SNP trn ()aTEM-1, trn ()aTEM-8 cng xut hin hai
base A mi tai hai vj tr SNP 162.1(AGT) v 26(AGT). Trong khi d, khng tm thy su thay di no tai hai vj tr SNP
cn lai l 162.2 v 182 hay cc base ny vn duoc bo tn ()aTEM-8 (hnh , dui). Do vy, tai hai vj tr SNP 162.1
v 26 trn ()aTEM-1, base C v G duoc thay th bng hai base A tai cc vj tr SNP tuong ng trn ()aTEM-8, hnh
thnh nn 2 codon mi v duoc vit thnh CGT(162.1)AGT v GGT(26)AGT. Su thay th ny cng dn dn thay di
ca thnh phn amino acid tuong ng l Arg(162)Ser v Gly(26)Ser v tao thnh bin th beta-lactamase mi vi tn
goi TEM-8 .
H:nh 38 Co P6nh c3`ng @a ;Kn hi2u huYnh UuFng ;Si c6c DFPb NI, &, T, O ;huac c6c LM ;9K CN- Hh6c nhFu ;9n *laTGMc11X Nh:nh
;9nO L *laTGMcd Nh:nh >3eiO. Cung d tn hiu cao nht ca mt trong bn base tai mi vj tr SNP goi l PM, cung d tn hiu tai
ba base thp hon cn lai goi l MM. Chun ha vic so snh cung d tn hiu gia cc base thng qua t s PMPM v MMPM. Gi trj
PMPM 1 ng vi lai bt cp hon ton tai base d, gi trj MMPM 1 ng vi lai bt cp khng hon ton tai cc base cn lai.
Hai bin th TEM-116 v TEM-8 l cc beta-lactamase ph rng (ESBL) du bt ngun t TEM-1 v duoc tm thy
nhiu $- co)i v K- pn&*onia&. Tuy nhin, khc vi TEM-1 ch yu khng penicillins, TEM-116 v TEM-8 c kh
nng khng lai c amoxicillin, mt s thuc nhm oxyimino-cephalosporin nhu cefotaxime, cefpodoxime, ceftazidime v
oxyimino-monobactam nhu aztreonam . Tnh khng khng sinh ph rng ca cc bin th ny l do thay di mt s
nucleotide (qu trnh dt bin) tai mt s codon dn dn thay di thnh phn v cu hnh amino acid trong trung tm
hoat dng ca chng. Chnh v th, cc phuong php pht hin v chn don phn t d pht trin vi muc dch pht
hin nhanh cc bin th ny thng qua vic xc djnh su thay di v trnh tu nucleotide. Mt trong d phi k dn cc
phuong php PCR. Phuong php PCR thng thung c uu dim d thao tc, nhung khng phn bit duoc beta-lactam
ph rng hoc ph hp v cc bin th ca TEM, SHV . Trong khi d, PCR-SSCP c uu dim l phn bit duoc mt s
bin th ca beta-lactamase, nhung cn cc diu kin cho qu trnh chay din di trn gel agarose phc tap cng phu .
Bn canh d, phuong php PCR-RFLP c uu dim d thao tc hon v c th pht hin su sai khc nucleotide. Tuy
nhin, su sai khc nucleotide dn dn thay di vj tr gii han v v vy gy kh khn cho vic phn tch tip theo . Gn
dy, nh su ra di ca DNA-Microarray d dem lai hiu qu cao trong vic pht hin v chn don, dc bit xc djnh
nhiu bin th c kh nng khng ph khng sinh beta-lactam rng hon nhu cc bin th ca TEM v ca TEM, SHV
v CTX-M hoc cc bin th ca OXA .
Trong nghin cu ny, DNA-Microarray duoc ch tao v s dung d xc djnh cc vj tr SNP trn hai bin th TEM-116
v TEM-8. Bn vj tr SNP mi tm duoc trn hai bin th ny ging bn vj tr tm duoc bng DNA-Microarray truc d
v cng tuong ng vi bn vj tr nucleotide bj thay th trn trnh tu ()aTEM-1 d cng b . Ngoi ra, thi gian d pht
hin cc bin th TEM bng DNA-Microarray s dung trong nghin cu ny v truc d l tuong duong nhau trong
khong , dn 4 h. So vi cc phuong php PCR (PCR-SSCP, RT-PCR, PCR-RFLP.) , phuong php Vitek ESBL
hoc gii trnh tu nucleotide . thi gian pht hin ca DNA-Microarray nhu vy chua th hin ht tnh uu vit ca n
nu nhu chi pht hin mt di tuong. Tuy nhin, trong trung hop pht hin song song nhiu di tuong cng mt lc
trong khong thi gian khng di, th DNA-Microarray dem lai hiu qu to hn v mt thi gian v nng sut phn tch.
Bi l, do qu trnh dt bin dn dn hnh thnh cc bin th beta-lactamase ph rng ca TEM, SHV, CTX-M khc
nhau v do duoc truyn qua plasmid nn cc bin th beta-lactamase ny duoc tm thy nhiu loi vi khun gy
bnh, lan rng sang nhiu quc gia khc nhau . bi th cho nn DNA-Microarray dng vai tr nhu mt trong nhng
cng cu hu hiu d pht hin v chn don nhanh, song song, dng thi cc bin th ny. Hon th na, DNA-
Microarray cn l cng cu trong gii trnh tu v xc djnh kiu gen ca tng di tuong phn tch v v vy gip cho vic
diu trj kjp thi, chnh xc.
Z[T )%_N
DNA-Microarray tao ra trong nghin cu ny buc du duoc dng d pht hin hai bin th ca TEM-1 l TEM-116 v
TEM-8 trong khong thi gian 4 h. Vic pht hin cc vj tr SNP mi hay xc djnh kiu gen ca hai bin th s l co s
d pht trin cc loai DNA-Microarray tip theo vi muc dch pht hin v chn don nhanh khng nhng cc bin th
TEM khc, m cn cc beta-lactamase ph rng hon nhu SHV, dc bit CTX-M.
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Arlet G, Brami G, Decre D, Flippo A, Gaillot O, Lagrange PH (1). Molecular characterisation by PCR-restriction fragment length
polymorphism of TEM beta-lactamases. .$/" /ic'o(io) L&%% 13g(2)T 20-208.
Birkett C, Ludlam HA, oodford N, Brown DF, Brown NM, Roberts MT (200). Real-time TaqMan PCR for rapid detection and typing of
genes encoding CTX-M extended-spectrum beta-lactamases. 0 /&, /ic'o(io) iX(1)T 2-.
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PRODUCTON OF DNA-MCROARRAY FOR RAPD DETECTON AND
DAGNOSS OF TEM BETA-LACTAMASES RESSTANT TO EXTENDED
SPECTRUM BETA-LACTAM ANTBOTCS
Bui VFn Ngoc
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, Nghibm Ngoc Minh
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, Nguybn HoFi Thu
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, Nguybn Huu juc
2
, Nguybn juc ThFng
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The e0tended spectum beta-lactamases C-DZ[sF poduced b% man% bacteial pathogens ae esistant to e0tended-spectum beta-
lactam antibiotics b% h%dol%\ing the ]-lactam ing and beaking the stuctue o^ antibiotics* _^ these -DZ[s ae T-+ beta-
lactamases* 9ucleotide substitutions in some codons lead to amino acid changes that altes con^iguation o^ active cente o^ beta-
lactamases esulting in ^omation o^ man% T-+ vaiants esistant to boade spectum beta-lactam antibiotics* Wn the pesent stud%'
.9:-+icoaa% `ee ^abicated ^o apid detection and diagnosis o^ t`o T-+ vaiants' namel% T-+-==> an^ T-+-?' b% means o^
identi^ication o^ single nucleotide pol%mophisms CD9EsF at ^ive positions in blaT-+-==> and blaT-+-? taget seAuences* Zased on
pe^ect match and mismatch bet`een oligonucleotide pobes on micoaa% and ^agments o^ taget .9: duing h%bidation' the
D9Es `ee detemined and nucleotide substitutions CmutationF o consevation `ee also elucidated* Wn the blaT-+-==> seAuence'
bases Q and R at D9E positions ?S and =?S `ee eplaced b% bases : and T' espectivel% that leads to amino acid substitutions at
ValC?SFWle and :laC=?SFVal' espectivel%* ao blaT-+-? gene' t`o bases R and Q at D9E positions =>S*= and SX> `ee substituted
b% t`o bases : that also esults in amino acid alteations at :gC=>SFDe and Ql%CSX>FDe' espectivel%*
K&59o',s: DNA-Microarray, extended spectrum beta-lactamases (ESBLs), ()aTEM, single nucleotide polymorphisms
(SNPs), perfect match and mismatch, cyanine
Author for corresspondence Tel 84-4-1 Email nghiemminh@ibt.ac.vn