BAO CAO T 6 M TAT - H O I NGHI KHOA HOC K* Nif M 4 0 . N A M 11975.20151 THANH I A P VlfN HAN LAM KHOA HOC VA CfiNG NGHf VlfT NAM'
THifr Kf CHLTNG N O I TAI TAI T 6 H0T> M d i TRONG P H A N CTNG MULTIPLEX PCR C H A N B O A N BACILLUS ANTHRACIS
Cinh Thj Thu Hdng', Nguyin Thdi Sffn', NghKm Ng9c M i n h '
'Hoc vlin Qudn y- Bi QuSc Phdng, Sd ISO Phdng Hung, Hd Bdng, Hd Ndi, hangdlnhblolSgmall.com 'Vlin Nghiin cdu hi gen, SSlS Hodng QuSc Vlit, Cdu Glay, Hd Nil, nahlemmlnhainrnr „„
Tbih cfy thiit: Chin dodn phdn hi, 6^ bi$t Id cdc phuong phdp dvra tidn PCR <H dugc ung dvmg rgng rdi trong nhilu Dnh vvrc nhu y hgc, mdi trudng hay khoa hgc hmh SVI... Tuy nhiln, mOt nhugc dilm Idn trong hiu hit cdc cdng bl ndy deu khdng dl edp den chdng ndi tp (mtemal amplification control - IC) - thdnh phin cin thiit vd cin nhu bit budc ttong PCR chin dodn. Myc dich cda chdng n9i tp Id xdc minh kit qud Chan dodn, hdnh hi?n tugng dm tinh gid, gdy sai sdt ttong chin dodn. Do dugc bl sung vdo mlu td qud trinh tdch chilt acid nucleic hay trong thvic hi#n PCR nSn chdng ndi tai cd thi kilm sodt dugc todn bd qud trinh chuin bj mlu vd thvic hi^n PCR (thiit bj ludn nhiSt, cdc thdnh phin trong master mix PCR hay chit dc chi cd trong Hmg mlu cu thi) tdy thuOc vdo thdi dilm sd dvmg.
Chdng ndi tp cd thi dugc chia thdnh hai lop dvra vdo nhu ciu sd dvmg mli, do Id chiing ndi tp cpih tianh (competitive IC - sd dvmg mli chung vdi cdp mil chin dodn) vd chdng ndi tp khdng cpih tranh (noneompetitively IC - sd dyng c§p mli rigne re, khdc bi$t vdi mli chin dodn). Trong dd, hudng thiit kl ehting ndi tp cpih tranh cd nhilu uu dilm hon, nhit Id ttong phdn dng mutiplex PCR (mPCR - PCR da mli) duoc khuyin cdo sd dvmg. Phuong phdp thudng dugc sd dvmg dl thiit kl chdng ndi tp canh tranh Id tdi tl hgp di truyin, t^o plasmid ehda dopi gen xdc dinh cho sdn phim PCR en kich thudc phdn bipt vdi band dich chin dodn. Trong cdng trinh ndy, mdt chdne ndi ta"
tdi tl hgp dugc thiet kl trong chin dodn chinh xdc BaciUus anthacis gdy b&ih d VSt Nam bdng mPCR Chdng ndi tai cpih tranh dugc thiit ke Iheo hudng tdng kich thudc sdn phim dich md vln sd dvmg chung mdt ttong cdc cdp moi chin doda Chiln luoc thiit kl chdng ndi tai ttong nghiin cdu ndy cung cd thi dugc dp dvmg ttong chin dodn chinh xdc cdc tdc nhdn vi sinh khdc dvra ttdn PCR.
Phtnmgphip nghiin cuu
Canh khuin 0,5 McFarland chdng B. anthacis Bai gdy benh luu gio tai Hoc vi"
Quan y dugc bit hop 121°C/20 phdt trudc khi tdch chilt DNA ting sl. Mdt ttinh tu DNA xdc dinh (dopi gen lef ehdng B. anthacis Bai- ICl) dugc khulch dp chinh xdc sd dvmg hda chat PCR high fideUty (Theimo scientific) vdi c ^ moi IClF-BamHI/R
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BAO CAO T 6 M TAT - H O I N G H I KHOA HOC K* Nif M 4 0 N A M (197S-201S) THAHH I A P Vlf N HAN I A M KHOA HOC VA CfiNG NGHf VlfT NAM'
NdeL Chung ndi tp dugc thiit kl qua cdc budc tdi t l hgp tdm tdt nhu sau: San phim PCR tinh sach ICl dugc t?o ddng phvi trong vector pJET1.2^1unt (Ihermo scientific) tjo plasmid pJET1.2-ICl. Sdn pham ICl dugc thu hii ijlng tmh s?ch gel (sau khi cit plasmid pJET1.2-ICl bdng c§p enzyme hpi chi BamHI vd Ndel), gdn vdo plasmid pIET1.2-capA (sau khi dugc cdt md vdng cdng bdng cdp enzyme hpi chi ndy). Qua qud trinh t?o ddng, plasmid pIET1.2-capA-ICl tao ra dugc nhdn bdn vdi sl lugng Idn ttong tl bdo £ coli DH5a. Plasmid tdi t l hgp ndy dugc kilm tta bing PCR khuin Ijc, cdt bdng enzyme hpi ehl vd gidi tiinh tvr. Chiing ndi tai plasmid pJET1.2-eapA-ICl dugc khuech dp ding ttidi trong phdn ling mPCR (chda ba c|ip moi vnAFl/Rl, pagAFl/Rl vd capAFl/Rl) chin dodn B. anthracis gay bfnh.
Tim tit kit qui vi thio lupi
Chdng ngi tp pJET1.2-capA-ICl dugc thiet ke trong chin dodn B. anthracis gay b^nh bdng phdn dng mPCR. Phan dng mPCR vdi 3 c$p mli Id virAFl/Rl, pagAFl/Rl vd capAFl/Rl dl xdc dinh svr cd m$t ei^ ede gen tuong iing d$c trung cho B.
anthracis gSyb^iiL Sdn phim mPCRcd kich thudc lin lugt E 222,751 vddlObpxdc dinh cdc gen tuong dng vrrA, pagA vd capA ciing vdi sdn phim khulch dai plasmid chiing n$i tp (sd dvmg chung cdp mli capAFl/Rl, cho sdn phim cd kich thudc 1000 bp) dl ddng phdn bi$t tidn ban dipn di agarose 1,5%.
Trong chin dodn vi smh vdt mau ldm sdng cdng nhu vi sinh v$t ngodi mdi trudng hay thvic phim bdng PCR, ehdng ngi tp Id rit cin Ihiit vd gin nhu bdt budc dl lop ttd hipn tugng dm tinh gid. Chdng ngi tp dd khdc phyc dugc nhiing hpi ehl cda chdng ngop (external control) - vdn dugc ddng phi bien. Do chdng ngop dugc bl sung vdo mdt dng phdn dng tdch bi§t (mdu ddi chdng), khuech dp song song vdi mlu chin dodn trong cdng dieu kiSn "«- *' m sodt dugc hidn tugng dm tinh gid trSn cdc
i khd nang kilm scat dugc thiit bj ludn nhi^
I chiing ngoai) Ihl chiing nOi tp cdn cd tbi lucleic vd cdc chat dc chi dnh hudng trong
i dugc thiet kl sd dyng chung cap moi B. anthracis gdy blnh. Tr6n co sd cd sdn lit cda c|p moi capAFl/Rl), chdng tdi dd cda mli chin dodn (264 bp) vd thay vdo on (chinh la dopi gen lef - ICl - 654 bp)
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BAO CAO TdM TAT ' H d l NGHI KHOA HOC K^ Nif M 40 N A M (197S-2015) T H A N H LAP VlfN HAN L A M KHOA HOC V A C A N G NGHE v i f r N A M *
tjo chdng ngi tp Id plasmid tdi tl hgp cd tin p]ET1.2-capA-ICl. Sy cd mjt cda chdng ndi t^ dugc bl sung vdo mlu ttong qud trinh tdch chilt DNA vdi lugng tii thilu vda dd cho phep xdc minh kit qud chin dodn, lop ttd hi|n tugng dm thih gid.
Tir khda: Am tinh gid; Chung ndi Idi; PCR chdn dodn; PCR da mSi; Tdi ti h(ip.
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