CHUYSN eg; HQI NOHI KHOA HQC HUYfff HOC • TRUYJW MAU TOAN QUgC M12
40001 thay cho phuong phip c i diin. Tuy nhifn, s i miu khio chua dd Idn di cd f nghia hon. Trong thdi gian sip tdi, chdng tdi sl tiip tpc nghifn cdu vdi s i mSu Idn hom di cd kit lufn chinh xic.
V. Kir LUf N
Phuong phip phin tich t i bio trong mft s i lofi dich CO thi bing miy Sysmex 40001 cho kit qui tin cfy dupe, thdi gian tti Idi kit qui nhanh v i khich quan, cd thi thay thi phuong phip c i diin.
TAI uf U T H A M KHAO
1. Brazdzlonyte, J. & Macas, A. (2007),
"Bland-Albnan analysis as an approach for statistical evaluation agreement between two methods {»
measuring hemodynamics during acute myocardial infaiction.", Medicina (Kauniu).
43,208-214.
M.H. dc Kcljzer, W.Van der Meer (2002) Automated counting of nucleated red biool cells
in blood samples of newborns. Clin. Lab.
Haem. 24,343-345.
Mary L.Turgcon, EdD, MT (ASCP) (2005), Body fluid analysis. Clinlcil Hematology Theory and Procedures, 5*
edition .25,465-495.
TRI^N KHAI QUY TRlNH Kf THUAT PCR DjNH LIIDN6 TRiN Td HOP GIEN MINOR BCR/ABL VA TEI/AML1 TAI seNH VllN TRUYiN M A U H U Y ^ HQC TP. Hd CHf MINH
T r i n Thj True Thanh', Phan Nguyin Thanh V i n ^ Pban Thj Xinb'
T6MTAT
Ky thuft PCR djnh lupng (RQ-PCR) l i phudng phip dupe uti tifn Ig^ chpn de dinh gli ton luu t l bio i c tinh trong bfnh bach (Su cap ddng lympho (BCCDL) cd bleu hlfn cic td hpp glen. Trong nghien cdu niy, chdng tdi thilt ifp dieu kifn RQ-PCR cho to hpp gien minor
BOVABL (mBCR/ABL) va Tm/AMLl, la 2 kilu t6 hpp glen thudng gfp nhat ttong BCCOL-B, ^ dgng dopn dd TaqMan. Ket qui cua nghien ciiu cho thiy dudng chuSn cua ABL, mBCR/M va TEL/AMLl da dupe xiy dung thanh cdng vdi R'>
0,995 v i hllu suit phin dhg tifn 95%. Vifc thif lap dieu kiln RQ-PCR cua mBCR/m. vi 'Khoa Dl truyen hpc Phan Tiy, Bfnh vifn Truyen mau-HuyS hpc TP.HCM.
^Khoa Mien Djch, Benh vien Truyen mau-Huyethgc TP.HCM.
^Bd mdn HuyS: hgc, Dal hgc YDuVc TP.HCM va Khoa Dl truyen hgc Phan Tu;
Benh vien Truyen mau-Huyethgc TP.HCM.
Phan lilfn khoa hpc: TS. Phaii Th| Xinh
Y HQC VIgT NAM THANQ 8 • stfflAC BI6T/2012
TEL/AMLl la kit qui bUdc dSu giup hoih thifn quy ttinh khao sit ffin luu t l bio i c tinh bdn BCCDL-B tfi Bfnh vifn Truyfn miu Huyit hpc TP.HCM.
SUMMARY
DEVELOPMENT OF REAL-TIME QUANTITATIVE PCR PROCEDURE ON MINOR BCR/ABL M«t TEL/AMLl FUSION GENE AT BLOOD TRANSFUSION
AND HEMATOLOGY HOSPITAL IN HOCHIMINH CITY
Realtime Quantitative Polymerase Chain Reaction (RQ-PCR) Is the first technique of choice for detection of minimal residual disease in acute lymphoblastic leukemia (ALL) expressed fusion genes. In this research, we established conditions for TaqMan-based RQ-PCR of minor BCR/ABL (mBCR/ABL) and TEL/AMLl which are tile most frequentiy occuning fusion genes in B- iineage ALL (B-ALL). The results of this study showed that the standard curves of ABL, mBCR/ABL, and TEL/AMLl were set up successfully witii R^>0.995, and PCR efficiency
>95%. Establishing conditions for RQ-PCR of mBCR/ABL aal TEL/AMLl ftision genes Is ttie first step In standardizing minimal residual disease procedure In B-ALL at Blood Transfusion Hematology Hospital, Ho Chi Minh City.
LOi^TV&NOE
Nhitag ky thuat di truyin hpc phin tfr nhu nhiem sic thi (NST) di, FISH (Fluorescent in situ hybridization) vd RT- PCR (reverse teanscriptase-polymerase chain reaction) l i cdng cy quan trpng ttong chan doan cic bit thudng NST va gien ttong cac bfnh mau ac tinh ndi chung va ttong bpch ciu cip ddng lympho (BCCDL) ndi rieng.
Kj* thuat RT-PCR cd dp nhay cao, va cho kit qua nhanh nhung chi dinh tinh ndn phd hop
cho vifc sing Ipc bit thudng mi khdng sd dpng di theo dSi dip dng diiu tr) [5]. Hifn nay, ky thuft ddng chiy ti bio (Flow cytometty) vi PCR dinh lupng (RQ-PCR) dupe xem li nhihig phuong phip dinh gii tin luu ti bio ic tinh hifu qui vdi tinh dfc hifu, dd nhfy cao vi cho kit qui nhanh [7,8].
Ky thuft RQ-PCR dupe uu tifn Ipa chpn tiding tnrdng hpp bfnh nhin cd mang cic ti hpp gien [4,7,8]. Trong BCCDL-B, t i hpp gien TEUAMLI vi minor BCR/ABL (mBCR/ABL) cd tin suit xuit hifn cao vdi TEUAMLI dupe tim thiy d 20% 30%
BCCDL-B ttd em [3,6] vi mBCR/ABL gfp ttong 20% - 30% BCCDL-B ngudi Idn [10].
Tfi Bfnh vifn Truyin miu Huyit hpc TP.HCM di khio sit thudng quy cic kiiu t i hpp gien thudng gfp ttong BCCDL-B bing ky thuft RT-PCR [l].Vifc ttiin khai quy trinh ky thuft RQ-PCR dii vdi 2 ti hpp gien TEUAMLI vi mmor BCR/ABL la budc ki tiip hoin thifn quy trinh chin doin cic bit thudng vi gien vi sd dpng cic bat thudng nay lim diu in gidp cho vifc theo ddi diiu tri.
n. Odi TUi;IN6 VA PHUONG PHAP NGHllN COU 2.L s i i tirpng nghiin cdn: Cic miu bfnh phim cua bfnh nhan BCCDL-B duong tinh vdi TEUAMLI va mBCR/ABL vi cac gien,4Bi, mBCR/ABL vi TEUAMLI.
2.2. Phirong phip tiin hinh 2.2.1. Khuich dpi gien ABL, mBCR/ABL va TEUAMLI
Tiin hinh khuich dfi gien ABL vdi mii xudi li QABL-F (5' GCC TCA GGG TCT GAG TGA AG 3') va mii ngupc la QABL-R (5- ACA CCA TTC CCC ATT G'TQ AT 3');
ti hpp gien mBCR/ABL vdi mii xudi la QBCR/ABL-mF (5' CTT CCA TGG AGA CGC AGA AG 3') vd mii ngupc la QBCR/ABL-mR (5' AAC GAA AAG GTT
121
CHUYgN eg: HOI NOHI KHOA HQC HUYgT HOC • TRUYJN M A U T O A N Q U 6 C 2012
GGG GTC AT 3'); vi ti hpp gien TEL/AMLl vdi mii xudi li QTEL-F (5' CTC TGT CTC CCC GCC TOA A 3") vi mii ngupc li QAML-R (5' CGO CTC GTO CTC OCA T 3') [5] sd dpng cic miu bfnh phim cda bfnh nhin duong tinh vdi TEUAMLI vi tnBCR/ABL. Polymerase ddng cho PCR li GoTaq (Promega, Hoa Ky). Kiim ba kich thudc bing phd hpp bing difn di ttfn thfch agarose 2% vi quan sit dudi tia UV.
2.2.2. Nil gien dich vio vector vi chuyin vector vio ti bio khi npp
Sin phim PCR dupe cho gin vio T- vector sd dpng bf kit pGEM-T-Easy vector system II (Promega, Hoa Ky) dieo hudng din cda nhi sin xuit Sau dd, vector mang gien dich dupe chuyin vio ti bio khi nfp JM109, ciy trin thfch LB borth cd chua ampicillin vi IPTG/X-Gal (Sigma, Hoa Ky) vi u d nhift dp37°Cttongl4-16gid.
2.2.3. Ly trich plasmid DNA va chuin bi mdu chuin
Thu nhin 5 - 7 khdm khuin Ifc U4ng, thpc hifn phin dng PCR kiim chin di chpn dupe khuan lac cd chua vector mang ddng gien quan tam vdi cfp mii li SP6-pGEM (5' ATT TAG GTG ACA CTA TAG AA 3') vi T7- pGEM (5' TAA TAC OAC TCA CTA TAG GG 3'). Sau dd, nhin ddng ti bio cd mang gien dich trong dung dich LB borth, ly ttich plasmid DNA bing bp kit QIAGIEN Plasmid Mini kit (QL\GIEN, Hoa Ky) theo hudng dan cua nhi sin xuit va do ning df plasmid DNA bing miy quang phi Ulttospec 5300 pro (GE Healthcare).
Dua vio ning dp vi ki'ch thudc cda plasmid
DNA, tiin hinh pha loSng miu dft ning df W bin saa/3^L, sau dd tiip tpc pha loing giim bfc 10 lifn tiip di cd dupe cic dung dicb plasmid DNA chda lO', lO', 10*, 1^, itf bin sao.
2.2.4. Thfc hifn phan ing RQ-PCR Cic lofi dofn dd duprc sd dpng ttong nghifn cdu li TaqMan Probe vdi diu 5' dupe dinh diu bing FAM vi diu 3' dupe dinh diu bing TAMRA. Phin dng khuich dfi cic ning df pha loing cda plasmid DNA td lO' din lO' bin sao dupe thpc hifn ttin miy CFX96 (Biorad, Hoa Ky).
III. KCT Q U A
3.1. Thilt Ifp diiu kifn chuin RQ-PCR Sau khi khio sit vdi cic ning df mii vi probe khic nhau, phin dng RQ-PCR vdi thi tich 20 pL cho kit qui tit d ning df probe li 260 nM vi ning df mii xudi vi mii ngupc diu li 900 nM. Chu ky luin nhift li 9S°C ttong 10 phdt vi ki tiip li SO chu ky gim 95°C ttong 10 giiy vi 60°C ttong 30 giiy.
3.2. Kit qui xiy dyng dudng chuin 3.2.1. Buing chuin cia gien ABL Dudng khuich dfi cda gien ABL cd dfng hlnh sigma gim 3 pha li pha tiim ting, pba log vi pha binh nguyfn (hinh IA). Dudng chuin dft tuyin tinh cao, vdi hf si hiong quan dft gii tr) >0,995, hifu suit phin dng dft >fl5% vi chu ky nguSng d df pha loing 10' bin sao cd gii tti hnng binh li 22,34 ± 0,46 (hinh IB).
Y HOC VlfT NAM T H A N O 8 • S 6 SAC BifT/2012
y IC copin—-'Id* I
u
Hlnh 1. Dudng khuich dfi vi dudng chuin cda gien ABL 3.2.2. Budng chuin cia ti hpp glen mBCR/ABL
Dudng khuich dpi cda mBCR/ABL cflng cd dfng hinh sigma gim 3 pha (hlnh 2A).
Dudng chuin dft tuyin tinh cao, vdi hf s i Wong quan dft gii trj >0,995, hifu suit phin dng dft >95% vi chu ky ngudng d dp pha loing lO' bin sao cd gid tti tinng binh li 23,02 ± 0,30 (hlnh2B).
B
uiaiMdl—>td5 - ^ t d 2
' " " • "
10
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" ' > * ' d 4 ^ ..Mi - I S -
Hlnh 2. Budng khuich dpi vi dudng chuin cua mBCR/ABL 3.2.3. Buing chuin cua ti hpp gien TEL/AMLl
Dudng khuich dai cda TEUAMLI cflng cd dang hinh sigma gim 03 pha (hlnh 3A).
Dudng chuin dft tiiyin tinh cao, vdi hf si hiang quan dft gia tti >0,995, hieu suit phin ung dft >95% vi chu ky ngudng d df pha loing lO' ban sao cd gii tti tiomg binh li 22,83 ± 0,92 (hinh3B).
Hinh 3. Dudng khuich dai va dudng chuan cda TEL/AMLl
123
CHUYEN Bi: Hpi NOH| KHOA HQC HUYtTT HpC • TRUYJN M A U T O A N QUgc 2012
3.3. Kit qui khio sit miu bfnh nhin:
Miu tdy xuong ldc chin doin cda 2 bfnh nhin BCCDL-B duong tinh vdi mBCR/ABL vi TEUAMLI dupe sd dpng cho phin dng RQ-PCR. Kit qui cho thiy chu ky nguSng ciia mii duong tinh vdi BCR/A Bi li22,56chuky, tuong dng khoing 10' bin sao (hinh 4A), vi chu kj ngudng cda miu duang tinh vdi TEUAMLI li 26,28 chu ky, tuong dng khoing 10'' bin sao (hlnh4B).
[ ,.
1 ! • 1
/,.W
Jl//I
(»•«
* *
Hinh 4. Dudng khuich dpi cda BCR/ABL vi TEL/AMLl
IV. B A N L U $ N
Nhift dp bit cfp mii tii uu chung cho phin dng PCR vd dieu kifn cua phin ung RQ-PCR khio sit chdng npi tpi ABL, ti hpp gien mBCR/ABL va TEUAMLI di dupe chuin hda nen ttong cung 1 phin dng PCR CO thi khuich dpi cung mpt luc ci 2 t i hpp gien, gidp tiit kiem thdi gian, hda chit va don gian hda qui trinh.
Dudng chuan cda chung npi tai vi miu chuin cua mBCR/ABL vi TEUAMLI dpt dp tuyin tinh va hifu suit phan dng cao. Dfc bift, dudng chuin cua gien ABL vdi hf si slope gin nhu dat gii tri ly Oiuyit -3,322 (ly thuyit li -3,32) va hifu suit phin dng li 100%. Dieu niy cho thiy di budc diu chuin hda dupe nong dp cac chit trong phin ung RQ-PCR. 6 dp pha loing lo' cua gien ABL, chu ky nguSng dat gii tri 22,34 ± 0,46 tuong duang vdi kit qua nghien cdu cua J Gabert va cpng su [5] li 22,30 d= 0,38. Kit qud khio sit mau bfnh nhin BCCDL-B cho biit dupe sd ban sao rit cao cua 2 ti hpp gien mBCR/ABL vii TEUAMLI luc chin doan, co
thi so sinh vdi miu thd sau cic giai dop diiu tri.
Ky tiiuft RQ-PCR cho kit qui ttpc tiip trfn miy mi khdng cin qua binJv difn di vira giup giim dfc hfi cho ngudi thao tdc va tiet kif m thdi giari. Budc diu chdng tdi di thinh cdng ttong vifc thiit Ifp diiu kifn cho kf tiiuft RQ-PCR su dpng dofn dd TaqMan ttfn ti hpp gien mBCR/ABL vi TEUAMLI iS tiin t(Sri dng dpng ttong dinh gii tin luu te bio ic tinh 2 td hpp gien niy trong tuong lai gin.
V. KiT L U A N
Day Ii nghien cdu diu tiln tfi Vift Nam khio sdt cdc diiu kifn xay dpng ky thuft RQ- PCR nhim thiit Ifp dudng chuin cho chdng npi tfi va dudng chuin cho td hpp gien TEUAMLI va mBCR/ABL giup dinh gia ton luu te bio ic tinh ttfn hai t i hpp gien niy d bfnh nhan BCCDL-B, gdp phin theo doi dip ung diiu tt) mpt each chinh xac, nhanh chdng, vi hifu qua.
Y HQC V I C T N A M T H A N Q B - s 6 P A C BlgT/2012
TAI U|U THAM KHAO
1. Phan Nguyin Thanh VSn, NguySn Ti^n Binh, NguySn Thj Kim D|nh, Phan Thj Xinh. Xkc dinh 4 th h(?p gen TEL/AMU, BCR/ABL, MLUAF4, E2A/PBX1 trong b?ich c^u cip ddng lympho t^\ b$nh vi§n Truyen Mfiu Huy^t Hoc TP. Hh Chi Minh. Y hpc TP.HCM201I;15(4):493-97.
2. Bao F, Munker R, Lowery C, et at.
"Comparison of FISH and quantitative RT- PCR for the diagnosis and foUow-up of BCR/ABL-positive leukemias". Moi DIagn 77ier2007;l 1(4): 239-45.
3. CayuelaJM^Banichel A,eraf.-TEL/AML1 fusion KNA as a new target to detect minimal residual disease in pediatric B-cell precursor acute lymphoblastic leukemia. Blood 1996;88:302-308.
4. Gabert J. Detection of recurrent translocations using real time PCR;
assessment of the technique for diagnosis and detection of minimal residual disease.
Haematohgical 1999;84(4):107-115.
5. Gabert J, Beilaird, et at. Standardization and quality control studies of "real-time"
quantitative reverse transcriptase polymerase chain reaction of fiision giene transcripts for residual disease detection in leukemia - A Europe Against Cancer Program. Leukemia
2003;17:2318-2357.
6. Golub TR, Barker G, et ai. Fusion of the TEL giene on 12pl3 to the AMLl giene on 21 q22 In acute lymphoblastic leukemia.
Proceedings of the National Academy of Sciences of the United State of America
1995;92:4917-4921.
7. Kerst G, Kreyenber H, et td. Concurrent detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia by flow cytometry and real-time PCR. British Journal of Haematology 2005;\2Z.774-7S2.
8. Neale G, Couatan-Smith E, et aL Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia. • Leukemia 2004;18:934-938.
9. Niels P, Neils C, et aL Rapid and Sensitive Minimal Residual Disease Detection in Acute Leukemia by Quantitative Real-Time RT-PCR Exempliflied by t(12;21) TEL/AMLl FusionTranscript, Genes Chromosomes. Cancer 1999;26:355-365,.
10. Pui CH, Crist WM. Biology and clinical significance of cytogienetic abnormalities in childhood acute lymphoblastic leukemia.
5/oo(/1990;76;1449-i463.
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