DI^N
OAN Y HOC Nghien cu'u khoa hocPHAT HIEN DOT BIEN itLlSOM VA rtM204I/V KHANG LAMIVUDINE a NHUlMG BENH NHAN VIEM GAN B MAN TINH BANG REAL-TIME PCR
Ho Thi Thanh Thuy*, Doan Mai Phu-ong**, Vfl Thi Tirdng Van"
TOM T A T
Muc tieu: su dung kT thudt real-time PCR diphdt hiin dot bien rtL180Mvd rtM204V/l khdng Lamivudine.
Bdi tuang vd phuang phdp: ba hi moi. mdu do sir dung trong nghiin cuu ndy dugc thiit ki dira vdo trinh tie cdc gene polymerase cua virus dugc cong bo trin ngdn hdng gene vd dugc tong hgp bdi cong ty IDT (USA). Sir dung 50 mdu huyit thanh ciia binh nhdn viem gan virus B mdn tinh da dugc diiu tri Lamivudine tgi Binh viin Bgch Mai vd Binh viin Dgi hgc Y dugc thdnh pho Ho Chi Minh. Sdn phdm PCR chira cdc vi tri dot biin rtL180Mvd rtM204I/Vdd dugc xdc dinh bdng phuang phdp real-time PCR sau do gui din cong ty Macrogen, Hdn Quoc di tinh sgch vd gidi trinh tu. Ket qud vd ket ludn: 11/50 mdu xudt hiin dot biin rtl80M, 9/50 mdu xudt hiin dot biin rt204Vvd 12/50 mdu xudt hiin dot biin rt204L Trong do. co 2 trudng hgp xudt hiin dong thdi hai dgt biin rtl80M vd rt204V, co 3 trudng hgp xudt hiin dong thdi hai dot biin rtl80Mvd rt204I, co 1 trudng hgp xudt hiin dong thdi hai dot biin rt204I vd rt204 V. Cdc kit qud Real-time PCR ndy da dugc kiim chimg lgi bdng gidi trinh tye sdn phdm PCR vd kit qud ciia cd hai phuang phdp cho thay hodn todn trung khdp trong phdt Men cdc dot biin.
Tu khda: dot bien khdng Lamivudine - viem gan vims B man tinh
I. DAT VAN DE
Lamivudine Id mdt trong ndm loai thudc da dugc FDA chdp thudn diing trong dieu tri viem gan vims B tu ndm 1998. Lamivudine, mdt ddng phan ctia nucleotide, vdi dich tdc ddng la en2yme reverse transcriptase ciia vims viem gan B (HBV) trong sudt qud trinh nhdn len cua vims. Sy khdng Lami'vudine bdt ddu xudt hien vd gia tang theo thdi gian, tir
16-32% (sau 1 nam dilu tri)^ len din 83% (sau 4 nam dilu tri). Nhiing dot biln da dugc ghi nhan Id nguyen nhdn chinh cua viec khdng Lamivudine bao gdm rtLlSOM va rtM204V/P'^ da dugc tim thdy trong viing chiic nang B vd C ctia gene ma hda cho enzyme reverse transcriptase ciia vims HBV. Cdc dot biln nay cd thl xdy ra dan le hodc kit hgp vdi nhau, vd cung cd thl xudt hien ciing vdi cdc dot biln
* Cong ty cSphdn Cong nglie Viet A
** Benli vien Bgcfi Mai
bd sung nhu rtV173L, rtV207I... Nhimg dot biln bd sung ndy dugc cho la giiip chiing khdng thich ting cao hon trong viec nhdn len '•^•^•'
Cd nhieu kT thudt sinh hgc phdn tu da dugc ung dyng de phdt hien cdc dot bien ndy. Dd Id cdc kl thudt gidi trinh ty, ki thudt PCR ket hgp lai phdn tu, PCR ket hgp viec dung enzyme cdt han che sau dd (PCR-RFLP), hay RFMP - restriction fragment mass polymorphism.. .''• ^'' Chiing tdi lya chgn ki thuat Real-time PCR nhdm phdt hien dot bien rtLlSOM vd rtM204V/I vi do chinh xdc, do nhay cao ctia ki thudt. Han nira. Real-time PCR cd khd nang phdt hien duge dot bien eho du nd chi chiem sd lugng nhd trong qudn the hdn hgp chua cd vims dang hoang dai vd dang dot bien. Vi vdy chiing tdi tien hdnh de tdi nham myc tieu: Sir dung kT thudt Real-time PCR diphdt hiin dot bien rtLlSOM vd rtM2 04 V/I khdng Lamivudine cua virus viem gan B.
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DIEN DAN Y HOC
Nghien cij'U khoa hoc
IL DOI TUONG VA PHUONG PHAP 1. Doi tirgrng
Mau bfnh ph^m vd amplicon: 50 mau huylt thanh benh nhan viem gan B man tinh dd vd dang dugc dilu trj bdng Lamivudine t^i B?nh vien Bach Mai vd Benh vien D^i hgc Y dugc
Thdnh ph6 Hd Chi Minh cung cdp.
M^i, mliu dd vd amplicon: ba h^ mdi, mau do (dugc tdm tdt trong bdng 1) su dyng trong nghien ciru ndy dugc thilt kl dya vdo trinh ty cdc gene polymerase ciia vims dugc cdng bd tren ngdn hang gene vd dugc tong hgp bdi cdng ty IDT (USA).
Ten rtl80F rtl80R rtlSOP rt204F rt204R rt204P
rt204I
rt204V
Chirc ndng Mdi xudi Mdi ngugc
Mau dd Mdi xudi Mdi ngugc
Mau dd
Mdi ngugc Mdi ngugc
Bdng I. Moi vd mdu db su d^ng Trinh tv
5'-TATTCCCATCCCATCATCYTC -3' 5' - CCACTAGTAAACTGAGGCTT* 3' 5'-FAM TGGGAGTGGGCCTCAGTCCGTTTCT
-TAMRA-3'
5'- CCTATGGGAGTGGGCCTC -3' 5'-GCC CCC AAT ACC ACA TCA TC -3' 5'-FAMCACTAGTAAACTGAGCCAAGAGAAACG
-TAMRA-3'
5'-CCC CCA ATA CCA CATC AT CA** 3' 5'- CCC CCA ATA CCA CAT CAT CG ** - 3'
5'-CCC CCA ATA CCA CAT CAT CT * * - 3 ' 5'-CCC AAT ACC ACA TCA TCC AC *** - 3 '
Chieu ddi (bp)
22 22 25 18 19 30
19
18
Kich thirdrc sdn phSm PCR(bp)
85
125
* Vi tri bat cap nucleotide dang dot bien tai codon rtl 80 ndm tren ddu 3' cua mdi xudi rtl SOR.
** Vi tri bat cap nucleotide dang dot bien tai codon rt204I nam tren ddu 3' ciia mdi xudi rt204I.
*** Vi tri bdt cap nucleotide dang dot bien tai codon rt204 ndm tren ddu 3' cua mdi ngugc rt204V.
2. Phtfong phap
Tach chiet DNA: DNA tir mau huylt thanh benh nhan dugc ly trich bang phuang phdp sir dyng phenol/chloroform, phdng theo phuang phdp cua Chomczynski & Sacchi (1987): 200|il huylt thanh dugc them vdo 900^1 Trizol, pH8. DNA sau dd dugc tiia bang Isopropanol vdi chdt trg tiia. DNA dugc giir trong dung dich TE IX (Tris-EDTA) vd bdo qudn d -20°C cho den khi su dyng.
Real-time PCR: phdn ung dugc thyc hien tren may Mxpro-Mx3005P (Stratagene) vdi chuang trinh nhiet bao gdm 95°C- 5' (1 chu ki) vd 40 chu ki lap lai vdi 95''C- 15", dO^C- 1' Thl tich hdn hgp phdn ling Id 50^1 bao gdm: Ix dung dich dem PCR, 100 nmol/L mdi va mau dd, 400nmol/L moi loai dATP,
dGTP, dCTP vd dTTP, 1.5 units hot-start Taq DNA polymerase, 3mmol/L MgCl,. Mdi dot biln dugc thyc hien trong mdi phdn umg rieng biet.
Giai trinh tyr: san phdm PCR chiia cdc vi tri dot biln rtLlSOM vd rtM204IA^ da dugc xdc dinh bdng phuang phdp real-time PCR sau dd gui din cdng ty Macrogen, Hdn Qudc Ah tinh sach vd gidi trinh ty.
III. KET QUA VA BAN LUAN 1. Dp ddc hifu cua moi vd mlu dd
Trong thyc nghiem kilm tra do dac hieu cua cac he mdi vd mdu dd, cac amplicon mang cac dot bien dugc thiet ke dya vdo trinh ty cdc gene polymerase ciia vims dugc cdng bd tren ngan hang gene va dugc tdng hgp bdi cdng ty IDT (USA) dugc su dung.
14 I Y HOC LAM SANG S6 62 (Thang 6/2011)
DIEN OAN Y HOC
Nghien cii'U khoa hoc
Ampliflcation Plots
•; y - >• • • ; • • • l ' - - < - - - - l - - - - < : • • • • • ! > ' ] ' • * ' . ^ ' ^ - " ; l i
t 4 • I
I . , .1, lUM.^-i.,
rtL180MMut rt M204V Mut rt M204I Mut r t r t L 1 8 0 M W T rt M204V W T
rt M204I W T
Amptftcation Plots
Bieu do 1. Kit qua Idtao sdt liha nang Ichuecit d^i cua moi vd mdu do
Bieu dd cho thay mdu HBV cdc dang dot bien (ki hieu Mut) cd tin hieu huynh quang vugt tren tin hieu nen, eac mdu HBV dang hoang dai (ki hieu WT) cd tin hieu huynh quang thdp hom tin hieu nen. Nhu vdy he primer-probe-amplicon hoat ddng hieu qud vd dac hieu cho cdc dgt biln rtLlSOM vd rtM204I/V.
2. D9 nhay cua phan ung
Bilu dd cho thdy ca ba dang dd thi bilu thi cho cdc dang dot biln rtl SOM, rt204V va rt204I deu cho tin hieu huynh quang vugt fren tin hieu nen tir mau cd ndng do amplicon 10' copy/ml vdi chu ki ngudng (Ct) gidm ddn din mdu cd ndng do 10^ copy/ml. Mdu cd ndng do 10 copy/ml thi cd tin hieu huynh quang thap hom tin hieu nen.
10'copples/ml 10°coppies/ml 10*copples/ml 10*coppies/ml 10 copples/ml
10^cappies/ml lo'coppies/ml
GHI CHCJ:
^ : rtL180MMut
« » a « : rt M204V Mut j g . ; g , ? ^ : rt M204I Mut
Bieu dd 2. Kit qud liftdo sdt dp nh^y cua phdn iing
3. Kit qua real-time PCR tren mlu benh pham
Quy trinh dugc thu nghiem tren 50 mdu benh phdm viem gan B man tinh da vd dang dieu tri Lamivudine.
Kit qua cho thdy cd 11/50 mdu xudt hien dot biln rtl SOM, 9/50 mdu xudt hien dot biln rt204V vd 12/50 mdu xudt hien dot biln rt204I. Trong do, cd 2 tmdng hgp xudt hien ddng thdi hai dot bien rtl SOM vd rt204V, cd 3 tmdng hgp xudt hien ddng thdi hai dot biln rtl SOM vd rt204I, cd 1 tmdng hgp xuat hien ddng thdi hai dot biln rt204I va rt204V. Cdc kit qud Real-time PCR ndy da dugc kiem chung lai bang gidi trinh ty sdn phdm PCR vd kit qua cua ca hai phuang phdp cho thdy hoan todn triing khdp trong phdt hien cdc dot biln.
IV. KET LUAN
Phuang phdp phat hien dot biln rtLl SOM va rtM204I/V bang ki thuat Real-time PCR xdy dyng hodn toan phii hgp trong phdt hien dot biln khdng thudc Lamivudine d HBV.
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DIEN OAN Y HOC
Nghien cu'u khoa hoc
TAI LIEU THAM KHAO
1. Anna Lindstro m, Jacob Odeberg,and Jan Albert.2004. "Pyrosequencing for Detection of Lamivudine-ResistantHepatitis B Virus ". JOURNAL
OF CLINICAL MICROB10LOGY.p4788-4795 2. Anna S. F. Lok Fabien Zoulim, Stephen Locamini, Alessandra Mangia, Grazia Niro, Hilde DecraemerGeert Maertens,5Frank Hulstaeri, Karen De Vreese,and Erwin Sablon 2002. "Monitoring Drug Resistance in Chronic Hepatitis B Vinis(HBV)-lnfected Patients during Lamivudine Therapy: Evaluation of PerformanceoflNNO-LiPAHBVDRAssay".JOURNAL OF CLINICAL MCROBIOLOGYp. 3729-3734
3. Chayama, K, Y. Suzuki, M. Kobayashi, M.
Kobayashi, A. Tsubota, M.Hashimoto, Y. Miyano, H Koike, M. Kobayashi, I. Koida, Y. Arose, S.Saitoh, N. Murashima, K Ikeda, and H. Kumada. 1998.
"Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lami-vudine
therapy and re-takeover by wild type after cessation of therapy " Hepatology 27:1711-1716.
4. Evelien Libbrecht, Joke Doutreloigne, Hilde Van De Velde, Man-Fung Yuen, Ching-Lung Lai, Fred Shapiro, and Erwin Sablon. "Evolution of Primary and Compensatory Lamivudine Resistance
Mutations in Chronic Hepatitis B Virus-Infected Patients during Long-Term Lamivudine Treatment, Assessed by a Line Probe Assay ". Joumal of clinical microbiology, Dec. 2007, p. 3935-3941.
5. Fiona Wightman, Tomos Walters, Anna Ay res, Scott Bowden, Angel ine Bartholomeusz, Daryl Lau, Stephen Locarnini, and Sharon R.
Lewin. Comparison of Sequence Analysis and a Novel Discriminatory Real-Time PCR Assay for Detection and Quantification of Lamivudine-
Resistant Hepatitis B Virus Strains, joumal of clinical microbiology, aug. 2004, p. 3809-3812
SUMMARY
DETECTION OF rtLl SOM AND rtM204I/V MUTATIONS ASSOCIATED TO LAMIVUDINE RESISTANCE IN SAMPLES FROM PATIENTS WITH CHRONIC HEPATITIS B VIRUS INFECTION BY REAL-TIME PCR
Objectives.' to develop real-time PCRs in order to detection of rtLlSOM and rtM204I/V mutations associated with resistance to lamivudine. Subject and method: we have established the real-time PCR methods using universal amplicons and taqman probes to detect rtLlSOM and rtM204I/V mutants by
individual reactions. The real-time PCR protocols were compared with sequencing and were applied on a further 50 clinical samples. Results and conclusions: the performance of these techniques were assessed by analyzing serial dilutions of amplicons of known concentrations and the lower limit of detection werefoundto be KPDNA copies/ml.
Evaluation of these techniques to detect lamivudine
resistance mutations was performed by analyzing 50 patients treated with lamivudine. 11/50 patients (for rtl SOM mutant), 9/50 patients (for rt204 V mutant), 12/50 patients (for rt204I mutant), 2/50 patients (for rtl SOM and rt204V mutant), 3/50 patients (for rtl SOM and rt204I mutant) and I patient (forrt204I/V mutant) were found. This result was confirmed by DNA sequencing. These real-time PCR protocols are rapid and accurate methods for quantification of rtl 81 rtLlSOM and rtM204I/V mutants of HBV virus in lamivudine-treatedpatients.
Key words: lamivudine resistance mutations - chronic hepatitis B.
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