Tap chi THONG TIN YDl/OC Sd 4 ndm 2010
(6.) Tiep tue md rdng tdi cac qudc gia thanh vien cac cd che nhu' Hpc vien Y te, nham nang cao nhan thifc sij'c khde va cac ldi sdng lanh manh thdng qua giao due dien tif (giao due dien tif d day dtfdc hieu la sif dung cdng nghe va phu'dng thifc dien tif ho trd cho qua trinh hpc tap);
(7.) Hd trd cho cac qudc gia thanh vien nham day manh su" phat then, ifng dung va quan ly cac tieu chuan qudc gia ve thdng tin y te; nham thu thap va ddi chieu thdng tin hien cd theo cac tieu
chuan de cd the thiet lap du'dc cac he thdng thdng tin y te chuan hda qudc gia, tao thuan ldi cho trao do'i thdng tin de dang va hieu qua giCfa cac qudc gia thanh vien;
(8.) Ho trd cho cac sang kien YTOT trong khu vi/c, lien khu vu'c hoac giifa cac nhdm/td' chifc d cac nu'dc-de cd tieng ndi chung.
CIMSI fNguon: Ninth plenary meeting, 25 May 2005,
Committee A, seventh report, www.who.int)
KY THUAT PCR VA UNG DUNG TRONG LAIVI SANG HUYET HOC
Nguyen Anh Trf, Vu Thi Bich Hu'dng"
Trong cac benh ly huyet hoc, cac rdi loan di truyen deu lien quan tri/c tiep tdi cd che sinh benh. De phat hien cac khiem khuyet di truyen do thi PCR va cac ky thuat cai tien tLr PCR du'dc coi la nhij'ng cong cu hang dau. Ngoai y nghia quan trgng de chan doan benh, phan loai benh phue vu dieu tri thi su" dung ky thuat PCR de phat hien gen benh ly con giup thdc day qua trinh nghien ed'u bao che cac loai thudc dieu tri nham dfeh de sCtdung trong y hoe.
I . G I O I THIEU CHUNG VE KY THUAT PCR PCR (polymerase chain reaction) la phan ifng td'ng hdp ADN trong dng nghiem, cho phep tach rieng mot gen rat nhd be tif mot he gen khd'ng Id va nhan ban mdt lu'dng Idn vdi dp chi'nh xac cao. Phan ifng PCR gdm cac thanh phan sau:
- ADN mach khudn: la viing ADN chifa trinh tl/ gen dfeh can nghien cifu.
- Cac dNTPs: gdm 4 loai deoxynudeotidetriphosphat la adenin, thymin, guanin va cytosine (day la cac ddn phan de hinh thanh nen ADN).
- Cac doan moi (primer): mdi la cac doan ADN ngan sdi ddn dai tif 20-30 nucleotid cd trinh ty' bo trd vdi trinh tt/ ciia hai dau doan ADN dfeh can nhan ban. Oe nhan ban mot doan gen can phai sif dung mdt cap mdi gdm cd moi xudi (forward primer) va moi ngLfde (reverse primer).
- Enzym polymerase chju nhiet: la enzym cd hoat tfnh td'ng hdp ADN, rat ben d nhiet do cao (enzym thu'dng du'dc sif dung la Taq- polymerase)
' PGS. TS., ThS., Vien Huyet hoc Truyen mau Trung u'dng
- Ion Mg^"" trong mudi MgClz d ndng dp mudi thi'ch hdp.
- Dung djeh dem de lam dung mdi trong phan ifng.
Khi dng nghiem vdi day dii eac thanh phan nhu' tren du'dc cho vao mot may luan nhiet (goi la may PCR), chi/dng trinh luan nhiet ciia may se lam cho nhiet do trong may thay ddi theo chu ky, nhd dd ma phan ifng nhan ban ADN sexay ra [1,7].
I I . NGUYEN TAC CUA PHAN U'NG PCR Phan ifng PCR gdm nhieu chu ky, moi chu ky gdm 3 du'dc cd ban:
- Bien tfnh (Denature, Melt): nang nhiet do len 94-96°C de ADN khudn tach thanh hai sdi ddn.
- Bat cap (Anneal); ha nhiet do xudng 40-60°C la nhiet do thi'ch hdp de cac doan mdi gan vao hai dau doan ADN can nhan.
- Td'ng hdp (Extent): nang nhiet do len 72°C, la nhiet do tdi Lfu cho su' hoat ddng ciia Taq-polymerase xiic tac hinh thanh cac mach bo sung.
Tgp chi THONG TIN YDl/OC So 4 nam 2010
ADN khuon
Moi xuoi
Bien tinh
«— Moi ngiidc „ ^ _ _ _ ^
_ Bat cap fong hdp
Chu ky dau tien
Chu ky thir 2
t^
-«*
Chu ky thif 3 Hinh 1: Minh hoa phan u'ng PCR
Nhu' vay, sau chu ky nhiet thif nhat, mdt ADN di'ch da du'dc nhan thanh hai ban sao. Sau chu ky thif hai, tif mdt AON di'ch ban dau se nhan du'dc 4 ban sao. Neu chu ky nay dtfdc lap lai 30- 40 chu ky thi tif mot ADN dich se nhan du'dc 2^°- 2 * ban sao (tife la hang ty ban sao).
I I I . CAC LOAI PHAN U'NG PCR
Cd nhieu loai phan ifng PCR. Ngu'di lam xet nghiem chon loai PCR nao la phu thudc vao muc di'ch cha'n doan va each thiet ke mdi sao cho cd the dat du'dc dp nhay va dp dae hieu nhat. Mot sd ky thuat PCR du'dc diing phd bien trong cha'n doan gdm:
1. PCR thdng thu'dng
La loai PCR du'dc phat minh dau tien (nam 1985), loai PCR nay sif dung mach khudn la ADN va mot cap mdi dae hieu de nhan ban (hay khuech dai) doan ADN mong mudn theo nguyen tac phan I I . Phu'dng phap PCR nay du'dc sif dung de phat hien cac vi sinh vat cd bd may di truyen la ADN (nhu' HBV, CMV) hoac nhan ban mot doan gen bat ky can nghien cifu.
2. PCR long (nested PCR)
La loai PCR phai tien hanh 2 vdng lien tiep va sif dung 2 cap mdi: cap mdi vdng ngoai va cap mdi vdng trong. PCR vdng 1 sif dung cap mdi vdng ngoai de nhan ban mot viing ADN, trong dd cd chifa doan ADN can tim, sau dd sif dung san pha'm PCR vdng ngoai nay lam khudn de thi/c hien phan ifng PCR vdng 2 (sif dung cap mdi vdng trong) de khuyech dai diing trinh t i / ADN can tim.
Nested PCR du'dc sif dung trong tru'dng hdp benh pham cd ft ban sao gen dich. Sau
phan ifng PCR vdng 1 khdng the phat hien du'dc san pha'm khuyech dai, do dd, phai nhd PCR vdng 2 lam giau gen di'ch dd len gap nhieu lan mdi cd the phat hien du'dc tren ban dien di. Ngoai ra, nested PCR cdn du'dc sif dung khi cap mdi thiet ke cho trinh t i / dfeh cd do nhay thap.
3. RT-PCR (Reverse transcriptase-PCR) La phu'dng phap PCR ma acid nucleic dich can phat hien ARN. Phan ifng RT-PCR bao gdm 2 du'dc la RT (sao chep ngu'pc) va PCR.
* Bu'dc 1: RT hay cdn gpi la du'dc sao chep ngu'pc, la giai doan ARN di'ch du'dc phien ma ngu'pc de trd thanh sdi ADN bd trd - cDNA (complement DNA). Day la giai doan quan trpng quyet djnh thanh cdng ciia phan ifng RT-PCR va du'dc thi/c hien bdi mdt loai enzym chuyen biet ten la reverse transcriptase, day la mdt loai enzym khdng chju nhiet va cd dae tfnh la sif dung mach ARN ddn lam sdi khudn de td'ng hdp nen sdi cONA.
* Bu'dc 2: sif dyng san pha'm cDNA d bu'dc 1 lam khudn cho phan ifng PCR tiep theo de nhan ban doan gen mong mudn.
Ky thuat RT-PCR phife tap hdn ky thuat PCR thdng thu'dng rat nhieu, vi tach ARN la mdt ky thuat rat khd, khd hdn nhieu so vdi ADN. ARN tdn tai d dang sdi ddn nen rat kem ben, trong khi enzym phan hiiy ARN (RNase) lai vd ciing ben vifng va cd mat d khap ndi (cd rat nhieu tren dau ngdn tay ciia ngu'di thao tac,...), do dd, phai tuan thii nghiem ngat ngay tif khau chua'n bj dung cu, phai chae chan rang dang thao tac tren cac dung cu da khif het RNase [3].
Tgp chi THONG TIN YDl/OC Sd 4 nam 2010
RT-PCR du'dc sif dung de phat hien cac vi sinh vat cd bd may di truyen la ARN (nhu' HIV, HCV) hoac de phan tfeh bieu hien gen.
IV. CAC BU'DC KY THUAT CHINH CUA XET NGHIEM CHAN OOAN BANG PCR
* Bu'dc 1: tach te bao lympho tif djeh tiiy hoac mau toan phan giau te bao bach cau.
* Bu'dc 2: tach chiet ADN/ARN tif te bao lympho.
Muc dich la de thu du'dc mdt lu'dng acid nucleic dii ldn va dii tinh sach lam nguyen lieu cho cac bu'dc tiep theo. Qua trinh tach chiet phai dam bao cac phan tif nay d trang thai nguyen ven tdi da (tranh dift gay). Do dd, trong qua trinh tach phai tranh cac thao tac cd hpc nhu' nghien, lac mau qua manh. Dung cu sif dung trong qua trinh tach mau phai du'dc khif het ONase (enzym phan hiiy ADN) va RNase. Qua trinh tach nen di/dc tien hanh d nhiet dp thap de ifc che cac enzym ndi bao (cac nuclease).
* Bu'dc 3: tien hanh ky thuat PCR, vdi cap mdi dae hieu de nhan ban doan gen dfeh.
Can chu y rang, cac thanh phan ciia phan ifng PCR can phai du'dc dat tren da trong sudt thdi gian mix mau. Rieng Taq-polymerase phai du'dc bao quan d tii am sau -20°C, chi lay ra sau khi da mix xong cac thanh phan khac va cat lai til am sau ngay sau khi sif dung. Ndng dp ADN/ARN cho vao moi dng phan ifng tif 10-lOOng.
Mo[ lan tien hanh xet nghiem PCR ngoai cac mau benh pha'm can xet nghiem thi phai ludn chay kem cac mau ddi chifng de dam bao chae chan ket qua thu du'dc la dang tin cay.
* Bu'dc 4: dien di san pham PCR.
Sau khi qua trinh chay PCR, ket thiic phai tien hanh dien di tren gel agarose de phat hien san pham PCR. Trong bu'dc nay, can chii y chpn ndng dp gel phii hdp vdi kfch thu'dc ciia san pham (vi du, san pham cd kfch thu'dc l-20kb thi chpn gel agarose 0,6-0,8%, 80-200bp thi chpn gel 1,5-2%,...). Thdi gian dien di cung phai dieu chinh theo kfch thu'dc san pha'm, san pham cd kfch thu'dc ldn thi can dien di trong khoang thdi gian lau hdn.
* Bu'dc 5: Dpc ket qua.
Trong qua trinh phan tfeh ket qua phai xem ddi chifng am va ddi chifng du'dng tru'dc. Cac ddi chifng nay phai cho ket qua chua'n xac thi ket qua xet nghiem dd mdi dii dp tin cay. Sau dd, di/a vao kfch thu'dc san pham theo tinh
toan ly thuyet (khi thiet ke mdi) va thang ADN chua'n (la hdn hdp gdm cac doan AND khac nhau ed kfch thu'dc da biet) de xac dinh bang san pha'm mong mudn.
1: Marker AND
2: Ddng te bao M3 nudi cay (ddi chifng du'dng) 3: [Ddng te bao am tfnh M3 nudi ca'y (ddi chirfig am) 4: Mau benh pham
5: H2O (ddi chifng am)
Hlnh 2: Dgc ket qua PCR tren gel agarose V. U'NG DUNG TRONG LAM SANG HUYET HOC
1. Trong benh Idxemi cap
Cac rdi loan di truyen gay ra benh Idxemi cap chii yeu la cac bat thu'dng ciia he gen tren NST, cu the la si/ sap xep lai gen mdt each ldn xdn (ma cac gen nay la cac gen quan trpng chi phdi qua trinh sinh tru'dng va biet hda ciia te bao) dan den qua trinh hinh thanh cac td hdp gen mdi. Cae to hdp gen nay bieu hien ra cac san pham protein mdi, chiing can thiep manh me vao qua trinh sinh trPdng va tru'dng thanh ciia cac te bao mdt each khdng diing lue (theo each ifc che hoac kich thi'ch phan chia) dan den tich tu va gay ung thu'. Vf du: gen lai AML/ETO trong chuyen doan t(8,21) (q22;q22) (d Idxemi cap the M2). Gen lai ABL/BCR p210kDa va pl90 kOa trong chuyen doan (9;22) (CML),... Cac chuyen doan nay rat nhd, khd ma cd the nhin thay du'dc tren nhiem sac the dd. Oe phat hien chiing thi can phai sif dung ky thuat RT-PCR vdi cac cap mdi dae hieu du'dc thiet ke d 2 dau doan gen lai. Khi trong mau xet nghiem cd cac rdi loan di truyen (tife la cd SLf xuat hien ciia cac td hdp gen lai) thi cac primer se tim den bat cap vao cac vj trf bd' trd tren to hdp gen lai dd va se nhan ban du'dc toan bd viing gen lai nay.
Lfu diem ciia ky thuat la phat hien cac to hdp gen lai nhanh chdng va chi'nh xac, vi chi khi cd mat gen lai thi mdi cd san pham PCR. Ddng thdi
T(ip chi THONG TIN Y Dl/OC Sd 4 mm 2010
ky thuat RT-PCR dat dp nhay cao, cd the chpn Ipc du'dc tdi da la 10"*^ va toi thieu la 10"^ tife la cd the phat hien du'dc it nhat 1 te bao benh ly trong 10^ te bao binh thu'dng, dap ifng du'dc nhu cau chan doan sdm de kjp thdi dieu trj hoac theo
ddi cac benh tdn du" tdi thieu [4].
Hien tai, Phdng Sinh hpe phan tif ciia Vien Huyet hpc Truyen mau TW da xet nghiem thu'dng qui phat hien mdt sd gen benh dae tru'ng cho cac benh mau thu'dng gap (bang 1).
Bang 1: Mot so gen benh dae tru'ng eho eae benh mau thu'dng gap
^'~~~~-~J[he benh Gen ^^~~~~-~^
BCR/ABL p210 BCR/ABL pl90 AMLl/ETO PML/RaRa CBFB-MYHll
LXMkinh ddng tuy
Cd lam Cd lam
LXM cap ddng lympho
Cd lam
LXM cap the Ml, M2, M4, MS
Cd lam
LXM cap the'MS
Cd lam
LXM cap the M4, M4eo
Cd lam Viec tim ra cac marker phan tif dae tru'ng
cho moi mdt benh ly la rat can thiet va cd y nghTa quan trpng de cha'n doan, tien lu'dng va dieu trii benh. Vdi muc di'ch md rdng pham vi chan doan de nang cao chat lu'dng dieu trj, Phdng Sinh hpc phan tif da va dang nghien cifu, dl/ kien nam 2010 se trien khai them mdt sd xet nghiem bao gdm:
- Xet nghiem phat hien dot bien JK2 (Janus kinase 2) bang ky thuat alien-specific PCR. Opt bien JAK2 V617F xuat hien vdi ty le tdi 90-95% d BN tang hdng cau vd can va du'dc coi la mdt trong hai tieu chua'n chi'nh de chan doan benh.
- Xet nghiem dinh lu'dng bieu hien gen (qPCR) de theo ddi qua trinh dieu trj benh ung thu" mau, cung nhu" phat hien benh tdn du' tdi thieu de ngan chan tai phat trong ung thLf.
2. Trong benh Hemophilia
Binh thu'dng gen ma hda yeu td VIII va IX nam tren nhiem sac the X cd nhiem vu san xuat yeu td VIII va IX de lam cho mau ddng.
Khi cac gen nay bj ton thu'dng thi hoac la lam giam chifc nang td'ng hdp hoac la khdng thu'c hien du'dc ehifc nang td'ng hdp dd nifa gay ra benh mau khd ddng. Theo cac nghien cifu khoa hpc da cdng bd, cac ton thu'dng nay ed lien quan den cac dot bien gen, trong dd dot bien diem la chii yeu (90%) [3]. Cae dot bien nay lam sai lech mdt vai nucleotid tren gen dan den mat vj tri nhan biet ciia mpt so enzym gidi han (RE) ma cac enzym nay vdn cd d ngu'di khde manh binh thu'dng. Vi vay, de phat hien ra cac dot bien nay thi ky thuat PCR du'dc thi/c hien nhu' the nao? Trong tru'dng hdp nay, ngu'di ta phai sif dung ky thuat PCR thdng thu'dng ket hdp vdi mdt ky thuat khac gpi
la FIPLF (restriction fragments length polymorphism).
RFLP hay edn gpi la phu'dng phap da hinh chieu dai cac doan cat gidi han, tife la ngu'di ta sif dung mpt enzym gidi han RE (restriction enzyme) la enzym cd kha nang nhan biet va cat tai nhCfng diem dae tru'ng de cat mpt doan ADN quan tam. Ooan ADN ban dau sau khi bj cat bdi enzym gidi han se tao thanh eac doan ADN cd kfch thu'dc nhd hdn. NhLf vay, sau khi tien hanh phan ifng PCR va RFLP, d mdi BN Hemophilia se cd sd lu'dng cac doan ADN cd the gidng nhau hoac khac nhau. Od chi'nh la su' da hinh thai ve chieu dai ciia cac doan ADN dae tru'ng d mdi BN. Can cif vao dd, ket hdp vdi cac mau ddi chifng (tif nhifng ngu'di khde manh binh thu'dng), ngu'di ta se xac djnh du'dc cac dot bien neu cd.
Vf du: trong benh Hemophilia A ed mdt dot bien kha dae truPg, dd la dot bien d vj trf nhan biet ciia enzym gidi han Bel I tai vung intron 18 (cd kich thu'dc la 142bp). O ngu'di khde manh binh thLfdng, tai viing intron 18 nay cd vj trf nhan biet ciia enzym gidi han Bel I va khi cat doan ADN dd vdi enzym gidi han nay se tao thanh hai doan ADN nhd hdn, cd kfch thu'dc la 43bp va 99bp. Nhu'ng d nhifng ngPdi bj dot bien tai vi tri nhan biet ciia enzym Bel I, enzym nay khdng the nhan biet du'dc vi tri eat eiia minh nen khi dem cat eung viing ADN dd vdi Bel I se khdng tao du'dc cac doan ADN nhd hdn ma van cdn nguyen doan ADN ban dau (142 bp) neu la nO' hoac nam gidi bi benh, hoac se tao thanh 3 doan ADN cd kfch thu'dc lan lu'dt la 142bp, 99 bp, 43bp neu la nif gidi mang gen benh (tife la mang dot bien di hdp tif) (minh hoa d hinh 3).
Tgp chi THONG TIN Y Dl/OC Sd 4 nam 2010
V'
142 bp -*- 1,5: San pha'm PCR nhan ban viing intron 18 d BN niJ va ngu'di khde manh binh thu'dng.
3,4: San pha'm PCR sau khi eat vdi Bel I eiia BN nu' va ngu'di binh thu'dng
2: Marker AND lOObp
142 bp 99 bp 42 bp
Hlnh 3: Ket qua PCR-RFLP the hien tren gel agarose Xet nghiem phat hien dot bien vj trf Bel I tai d
intron 18, vj trf Hindlll tai intron 19 hien da du'dc trien khai thu'c hien thu'dng quy tai Vien Huyet hpc Truyen mau TW. Trong thdi gian tdi, nhdm nghien cifu se tiep tue trien khai them xet nghiem phat hien dao doan intron 22. Day la dot bien lam pha hiiy hoan toan cau triic gen VIII gay ra Hemophilia A the nang, 40-50% BN Hemophilia A the nang cd mang dot bien nay.
3. Benh Thalassemia
Thalassemia la nhdm benh do rdi loan td'ng hdp chudi globin do ton thu'dng NST 16 hoac 11 tao nen cae HST bat thu'dng. Opt bien d Thalassemia cd the la cae dot bien thay the, dot
bien chuyen doan hoac mat doan. Ngu'di ta da phat hien du'dc rat nhieu dot bien d Thalassemia.
Rieng d beta thalassemia da cd gan 60 dot bien khac nhau [2]. Hien tai, Phdng Sinh hpc phan tif, Vien Huyet hpc Truyen mau TW da du'a vao xet nghiem thu'dng quy mpt sd dot bien thu'dng gap d Thalassemia nhtf: dot bien d codon 17, IVSII- 654, codon 41/42, codon 71/72, codon 28, Codon 29 bang phu'dng phap PCR. Sap tdi, chiing tdi se trien khai phu'dng phap Multiplex PCR de eiing lue phat hien dtfdc nhieu dot bien.
V I . KET LUAN
Su' phat trien ciia khoa hpc va cdng nghe da dem den cai nhin td'ng the va eu the hdn ve bife tranh sinh hpc trong cd the con ngu'di.
Nguyen nhan gay ung thtf eiing cac cd ehe sinh benh cung da du'dc hieu biet tydng tan
hdn. Trong cac benh ly huyet hpc, cac rdi loan di truyen deu lien quan tri/c tiep tdi cd ehe sinh benh. Oe phat hien eac khiem khuyet di truyen dd thi PCR va cac ky thuat cai tien tif PCR du'dc coi la nhifng cdng cu hang dau.
Ngoai y nghTa quan trpng de ehan doan benh, phan loai benh phue vu dieu tri thi sif dung ky thuat PCR de phat hien gen benh ly cdn giiip thiic day qua trinh nghien cifu che tao ra cac loai thudc dieu tri nham dfeh de sif dung trong y hpc.
TAI LIEU THAM KHAO
1. Ho Huynh Thijy Du'dng: Sinh hpc phan tif. Nha Xuat ban Giao due, 2002.
2. Bandyopadhyay A., Bandyopadhyay S., Chowdhury D.M., Dasgupta B. U.: Major Beta- Globin Gene Mutations in Eastern India and Their Associated Haplotypes. Hum Hered 1999;49:232- 235 (DOI: 10.1159/000022880).
3. Bowen D.3.: Haemophilia A and Haemophilia B:
molecular insights. Mol.Pothol.55: 1-8. Catalog and technique Reference 2005-2006. New England Biolab, 2002
4. Dongen V.3.3.M. et al: Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberration in acute leukemia for detection of minimal residual disease. Leukemia, 1999, 13, pp. 1901-1928.
5. Forbes B.A., Sahm D.F., Weissfeld A.S.: Bailey and Scott's Diagnostic Microbiology. Mosby, 2002.
6. Innis M.A., Gelfand D.H. Sninsky J.J.: PCR Application protocols for Functional Genomics.
Academic Press, 1999.
7. Rolfs A., Schuller I., Finckh U., Rofls I.W.:
PCR: clinical Diagnostics and research. Springer Verlag Berlin Heidelberg, 1992.0
