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Bao Cao
Bao Cao
H:nh 18 6ch DJ ;9K LM ;9K c6c m=u >? NFO PQ @3Ac >R5 ;9n 5hi"n HKnh @B ;So ;hnh mic9oF99Fy NDO. Mi vj tr SNP cn pht
hin s duoc dp lp lai 3 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 36 (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 dng 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
NDO. 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 Cy3 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 b tuong ng vi cch b tr v thit k trn hnh 1a.
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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 5 vj tr SNP cn xc djnh trn ()aTEM-1 l 8(GTT),
16.1(CGT), 16.(CGT), 18(GCA) v 36(GGT) cho thy:
i vi ()aTEM-116 (hnh 3, pha trn) tai vj tr SNP 8, 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 8 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(8)ATT. Trong khi d, tai SNP
16.1, 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 16.1 hay codon 16 ca ()aTEM-116 vn
duoc bo tn l CGT. Ging nhu SNP 16.1, SNP 16.(CGT) trn ()aTEM-116 vn duoc bo tn. Tuy nhin, tai SNP
18 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(18)GTA. Tri lai, khng c su thay th base no xy ra tai SNP
36 (hnh 3, 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 8 v 18 tao thnh hai codon mi tuong ng vi hai vj tr thay th l GTT(8)ATT v GCA(18)GTA.
Su thay th ny dn ti su thay di thnh phn amino acid tuong ng l Val(8)le v Ala(18)Val v qua d tao thnh
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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 16.1(AGT) v 36(AGT). Trong khi d, khng tm thy su thay di no tai hai vj tr SNP
cn lai l 16. v 18 hay cc base ny vn duoc bo tn ()aTEM-8 (hnh 3, dui). Do vy, tai hai vj tr SNP 16.1
v 36 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 codon mi v duoc vit thnh CGT(16.1)AGT v GGT(36)AGT. Su thay th ny cng dn dn thay di
ca thnh phn amino acid tuong ng l Arg(16)Ser v Gly(36)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 mc 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 dng 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 dng trong nghin cu ny v truc d l tuong duong nhau
trong khong 3,5 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 c 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 c 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.
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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 mc 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 (1995). Molecular characterisation by PCR-restriction fragment length
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polymorphism of TEM beta-lactamases. .$/" /ic'o(io) L&%% 13g()T 03-08.
Birkett C, Ludlam HA, oodford N, Bron DF, Bron NM, Roberts MT (007). 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 5-55.
Bradford PA (001). Extended-spectrum beta-lactamases in the 1st century: characterization, epidemiology, and detection of this
important resistance threat. C)in /ic'o(io) 1&v 1g(4)T 933-951.
(Khng s dng qu 1 Ti liu tham kho)
PRODUCTON OF DNA-MCROARRAY FOR RAPD DETECTON AND
DAGNOSS OF TEM BETA-LACTAMASES RESSTANT TO EXTENDED
SPECTRUM BETA-LACTAM ANTBOTCS
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