Bước đầu xây dựng quy trình PCR nhằm phát hiện thành phần động vật trong thực phẩm chay dựa trê vùng 16s rDNA ty thể

Gicii thwang SV.NCKH-Eureka ldn thti 15 nam 2013

Ky yeru khoa hoc

VIRUS GAY BENH VIEM GAN SIEU VI C: TAI XAC DINH KIEU GEN
TREN VUNG TRINH TIINS5B VA XAC DINH CAC DOT BIEN DA HINH
DON
NUCLEOTIDE (SNP) TREN GEN IL28B NHAM TIEN LYONG PHUONG PHAP
DIEU TRI THICH HOP CHO BENH NHAN
Le Huyn Ai Thay*, Tic Thi Ngpc Ming Dung, Thi Toy, Nguyin Virang Minh Hoang,
Triin Thi Diim Kau, Nguyin Thi Minh Hien.
Khoa Cong nghe Sinh hoc, Dai hoc MO; TP. HCM
2Trung tam than clocin Y khoa Medic, TP. HCM
3Cong ty co phan Cong nghe Viet A
De tai act giai Nhi Gal tinging Sinh vien nghien ctiu khoa hoc — Eureka lciit XV ncim 2013

TOM TAT
Viem gan Met, vi C la do virus Hepatitis C (HCV) gay ra, lam inh Inning nghiem trong den sire khoe con
ngtrai. NM-mg tong trinh nghien elm On day ghi nhan co sy lien quan gift kiEu gen, (genotype) va phan tuYp
(subtype) dm HCV vai dinhen benh va kha Wang dap img dieu tri doi yen timg phac do rieng. Viing gen ma hi:5a
protein khong eau true (Non- structural NS5B), RNA polymerase phi thuec RNA, vai * diem trinh to to ra phit
lyyp cho tong tac phan biet kieu gen va phan tuSrp HCV.
Muc tieu nghien ciru cua chi:mg toi, truac het la xay dung ca so du lieu cyc be clang tin cay ve kieu gen va
phan tuSrp HCV. Thuc nghiem duce hen hanh duo ten phuong phip PCR- giii trinh hr met phan Wing NS5B cita
cac dung virus tir cac mau benh pham mau va phan tich phi he phan tir de xac dinh kieu gen va phan tuyp HCV.
Doi von 12 mau benh pham, san pham giai trinh tinYen viing NS5B duct dua vao phan tich pha he phan tir va
cho ket qua phan biet kieu gen, phan tujrp voi gia tri bootstrap >90% a m6i phan nh6m: nhom 1 a (2 mau), lb (1
mau), 2m (4 mau), 6a (2 mau), 6e (2 mau); 6t (1 matt). Phuong phap ducyc xay dung nay to ra uu the han khi so
sanh von phuong phap real-time PCR dtra tren yang 5'UTR. Vi vay, nghien euu nay se dirge ma reng vai ca mau
Ian hon trong thai gian sap tEri. ,
Thong ke du lieu ve tan so xuat hien cac kieu gen nhiem HCV dac trimg theo dia ly, khu vut, cac kieu det bien

da hinh, don nucleotide ten gen IL28B trong cac khao sat thyc nghiem cua cac nghien ciru trtrot day, danh gia co
hay khong cac tinh chat quan trong (kieu gen not trei theo timg khu vtrc dia ly, cac kieu det bien SNP mang tinh
dac hieu dia phuong...). Thiet ke be mei dac hieu cho phan img giai trinh to (sequencing) vung NS5B, vimg gin gen
IL28B de do tim dot bien.
Tu. khoa: HCV, NS5B, phci he phcin tu, PCR- gidi trinh hi, RT-PCR.
MO DAU

Hepatitis virus HCV la nguyen nhan chinh gay ra benh viem gan sieu vi C, la moi de doa hang du v&' sire khoe
doi yen dan cu ten toan the giEri. Viet Nam la met trong nhfmg nu& co tjr le mac benh viem gan sieu vi C cao teen
the glen Pham DA, Leuangwutiwong P, Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S,
Theamboonlers A Poovorawan Y(2009) High Prevalence of Hepatitis C Virus Genotype 6 in Vietnam Asian.
Asian Pac J Allergy lmmunol,27(2-3): 153-160.).
Viec xac dinh kieu gen (genotype), phan tuSfp (subtype) virus HCV la eat quan trong (Lu et al., 2007; Baclig et
2010). Da, c biet moi ki4u gen khac nhau se cho phae do dieu tri khac nhau, chang han nhu loai thuee, thai gian
dieu tri, qua trinh dinbenh,... (Tokita H, Okamoto H, lizuka H, Kishimoto J, Tsuda F, Lesmana LA,
Miyakawa Y, Mayumi M (1996) Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes
in the second (2e and 2f), tenth (10a) and eleventh (11 a) genetic groups. J Gen Virol, 77(2): 293-301.Error!
Reference source not found.Error! Reference source not found.. Cac phuong phap phan tich kieu gen HCV
thirimg duce sir dung la phuong phap lai Innolipa, phuong phip giii trinh tin viing trinh to khong dich ma (5'-non
untranslated region-5'-UTR) hoac phuong phap Real-time PCR,...wv (Lu et at., 2007; Murphy et at., 2007; Baclig
MO, Chan VF, Ramos JD, Gopez-Cervantes J, Natividad FF (2010) Correlation of the 5' untranslated region
(5'UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping. Int J Mol Epidemiol
134


Ky yen khoa hoc

Giai thuring SV.NCKH-Eureka lan ilia. 15 nam 2013

Genet, 1(3): 236-244.Xia X, Zhao W, Tee KK, Feng Y, Takebe Y, Li Q, Pybus OG, Lu L (2008) Complete genome

sequencing and phylogenetic analysis of HCV isolates from China reveals a new subtype, designated 6u. J Med
Virol, 80(10): 1740-1746.0 Viet Nam, kieu gen HCV ph6 bien la 1, 2 va 6 (Pham DA, Leuangwutiwong P,
Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S, Theamboonlers A, Poovorawan Y(2009) High
Prevalence of Hepatitis C Virus Genotype 6 in Vietnam Asian. Asian Pac J Allergy Immunol,27(2-3): 153160.Pham DA, Leuangwutiwong P, Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S,
Theamboonlers A, Poovorawan Y(2009) High Prevalence of Hepatitis C Virus Genotype 6 in Vietnam Asian.
Asian Pac J Allergy Immuno1,27(2-3): 153-160.; Tanimoto T, Nguyen HC, Ishizaki A, Chung PT, Hoang TT,
Nguyen VT, Kageyama S, Oka S, Pham VT, Ichimura H (2010) Multiple routes of hepatitis C virus transmission
among injection drug users in Hai Phong, Northern Vietnam. J Med Virol, 82(8): 1355-1363.). Theo cac nghien city
truerc day, giai trinh t r vung gen kitting ma hoa a du 5' (5'NC 5' Non Coding) la thigh hop va da duoc ap,clong
khac cho rang dinh kieu gen tren vung 5'UTR se cho ket qua
trong lam sang. Tuy nhien, gin day mOt so nghien
khong chinh xac so vai viec sir dung viing gen dich ma Ma protein kh6ng eau true 5B (non structural - NS5B)
(Nakatani SM, Santos CA, Riediger IN, Krieger MA, Duarte CA, Do Carmo Debur M, Carrilho FJ, Ono SK (2011)
Comparative performance evalution of Hepatiris C virus genotyping based on the 5' untranslated region versus
partial sequencing of the NS5B region of Brazilian patient with chronic hepatitis C. Virol J, 8: 459.. Lj do la vung
5'NC c6 the nham Ian trong viec phan biet gifra kieu gen 6 voi phan tujp I b hay giAa 1 a va lb. Trong khi do, vung
NS5B voi nhang tinh chat ve trinh tu mang tinh bien Ong phi hop de phan biet giCia cac kieu gen va phan tujp tot
nen duoc khuyen cao dung de thay the cho vung 5'UTR.
Gen interleukin-28B (IL28B) la mOt gen da hinh. Gen ma hOa cho interferon loci I, ram tren nhiem sac the s6
19 dia ngtrai, a vi tri 19q13. Chinh do sir da hinh cila gen IL28B nen mot so cac bien the da hinh dO da sira dOi viec
sin xuat cac interferon do da anh huemg den dap Ung mien dich cua vat chit. Gan day, mot s6 cac bien the da hinh
gin gen nay gang da duoc thing minh lien quan den viec kiem sok berth viem gan C, va tien doan kha [fang dap
img diet' tri cua benh nhan virus C khi dieu tri vai PEG-INF/RBV.05 3 SNP dm IL28B dA duoc xac dinh, do la
SNP rs12979860, SNP rs8099917, va SNP rs12980275. SNP rs12979860 chinh la cong tic interferon: bien the C/C
la ding tic bat (switch-on) va C/T la ding tic tit (switch-off). Bien the C/C cho phep tien doan hieu qua dieu tri
thanh cong IFN cao (tren 80%) vial it nguy co tai phat va tien trien man tinh hon so voi Wen the C/T tien doin hieu
qua dieu tri that, voi nguy co tai Oat va tien trien man tinh cao.(Pham et al,.)
Muc tieu ctia nghien eau nay, truot het nham xay dkmg co so du lieu cue be) cho cac kieu gen IA phan tujp
HCV. Day la cong viec thiet thuc, lam co so cho viec so sanh, suy ra kieu gen, phan tujfp se duoc ap dung sau nay.
Phtrcrng phap RT-PCR ket hop voi giai trinh tu vung gen dich thus c vung NS5B dirge thiet lap de to do trinh to giai

duoc se &rot dung de phan tich phi he nham xac dinh kieu gen va phan tuyp HCV co trong mot so mau benh phim
thu nhan to Trung tam chan doan Y khoa Medic, TP. HCM.Btrac dau khao sat in silico vai trb cila SNP rs12979860
va rs8099917 doi vai dap Cmg dieu tri cua benh nhan viem gan C khi dieu tri voi PEG-IFNa/RBV tren the giai va 6
Viet Nam. Thiet ke va did nghiem kha nang hoot clOng ctla moi, he m6i (mau do) cho phan Ung Realtime-PCR
nham xac dinh 2 SNP quan tam.

NGUYEN LIEU VA PHIAING PHAP
Nguyen lieu
Cac matt benh pham thuOc 12 benh nhan bi viem gan C (duct cung cap bEri Trung tam chin doan Y khoa
Medic, TP. HCM).
Phwang phap

Xciy clung ca sa du lieu cuc 17 6. ye trinh ter vung NS5B virus HCV
Dog tren co so da lieu ngan hang gen NCBI va Los Alamos Laboratory va ter nguan cac bai bao tham khao, co
so du lieu cuc bo la tap hop nhAng trinh tu tham chieu thuOc viing NS5B.va trinh loci GBV-C/HGV lam nh6m
ngoai (outgroup) da duoc tap hop. Sau d6, thing toi nen hanh thong,ke so luting va phan yang dia ljt cho cac kieu
gen va phan tujp HCV (vu tien chon cac trinh tu tir HCV co nguon goc phan lop tai Viet Nam).
Khoo sat in silico ve 2 SNP rs12979860 va rs8099917

135


Giai thwerng SV.NCKH-Eureka Idn tit& 15 nam 2013

Ky ydu khoa hoc

Tien hanh khai thic dv lieu ten PubMed ve 2 SNP rs12979860 va rs8099917. Ding tiled tien hanh thOng
danh gia m6i quan he gicra cac loaf bien the veri chimg tOc, dia ly, dap ting dieu tri va kieu gen cua virus HCV nhiZm
tren benh nhfin.
Tach chill RNA

RNA duos tach chiet ter 12 mAu benh phfirn mau thuOc 12 benh nhan bi benh viem gan C bang phtrong phap
mu dung phenol/chloroform, phong theo phuong phap cua Chomczynski va Sacchi (1987): 200 pl huyet thanh duct
them vao 900 pl Trizol, pH4. Nucleic acid sau de &rot trig bang isopropanol veri chfit try trig GlycoBlue. Nucleic
acid thu dirge sau trig duct gii trong dung dich TE 1 X (Tris-EDTA) (deii v6i DNA) va H2O cat da qua xir ly bang
DEPC (dOi voi RNA) vabio quirt a -20°C cho den khi sir dung.
Tong hop cDNA va khuech clgi vimg gen NS5B
Thus. hien phin ling Reverse transcriptase PCR voi bO kit RT-PCR cim ding ty co phfin cong nghe Viet A
nham khuech dai cac ban sao cDNA 60 gen virus HCV (khoang 9.6 kb HCV) vai cac mau ngau nhien hexamer veri
oligo d(A) (20 nucleotide). Chuang trinh luan nhiet tong hop cDNA: 25°C trong 5 phin, 42°C trong 30 phut, 85°C•
trong 5 phtit, lam mat a 4°C. Sau do, cac san pham cDNA duot thuc hien phan ting khuech dai yang gen NS5B veri
cap mOi NS5B2F/NS5B2R tir ding trinh nghien ciru ctia tat gia Nakatani va dOng tic gii (2011): NS5B2F (5'TTCACGGAGGCTATGACYAGG), NS5B2R(5'- CGGGCATGMGACASGCTGTGA), cho san phim co kith
think 688 bpveri Wing the tich phan ling PCR là 25 pl theo chtrcrng trinh Juan nhiet phin (mg PCR: a 95°C trong 5
phitt; ti6p theo là 35 chu ky voi 94°C trong 2 phut, 50°C trong I phin, 72°C trong 2 phin; giai (Joan keo dai cu6i ming
6 72°C trong 10 phin va btrerc lam mat a 4°C. Cac san pham khuech dai vimg NS5B &roc gin di giii trinh tu er cOng
ty Macrogen (Hin QuOc).
Phan tich ket giai trinh to
Cac ket qui giai trinh tu vimg NS5B cua cac dong virus HCV &roc hieu chinh (proof-reading) va so sanh voi
hai ngutm co ser dir lieu: (1) ten ngan hang gen NCBI, (2): co ser du lieu cue be): Tir do, chung toi chon loc ket qua
va tien hanh phfin tich su phit sinh loci ctia virus HCV c6 trong 12 mail benh phfim voi cac phuong phap Neighborjoining (NJ), Maximum likelihood (ML) va Maximum Parsimony (MP), sir dung trinh tu tham chieu tir co so dir
lieu cue 13O not tren.
Khoo sat sy. hoat &Ong caa m6i khuech dal yang trinh to chat! SNP tren thyr nghiem
Tien hanh kiem tra su hoat Ong cim 2 cap m6i da chon bang phuong phap PCR ved chimg duong la mau benh
pham cim benh nhan viem gan C &roc cung cap bed Trung tam y. khoa Medic. Sau do tien hanh tip cac thi nghiem
khao sat nhiet dO lai toi tru
cap mei va dr) dac hieu
san phfim khue'ch dai.
KET QUA VA THAO LUAN

Xfiy dung err so' du. lieu cue b6 ve trinh ty. yang NS5B virus HCV
Qua phan tich chi tiet tren may tinh, chung toi ghi nhfin veng NS5B vira mang tinh bao ton lai c6 tinh bien

Ong rat phi, hop de phan biet de clang cac phan tuyp HCV so voi sir dung Wing 5' khong dich ma (Baclig et al.,
2010; Nakatani et al., 2011). Cac trinh to v6ng NS5B (lucre thu thap nhieu chung a nhieu vimg dia ly khic nhau
(chau Au, chau A, chau Phi..) (Tokita et al., 1994; Tokita et al., 1996; Lu et al., 2007; Pham et al., 2009; Xia et al.,
2008) dac biet co ser do lieu bao ham cac chung virus co ngu6n g6c phan lap a Viet Nam. DM vai kieu gen 1, chimg
tbi thu dirge 39 trinh voi cac phan tuyp tir la den 11. Kieu gen 2 co 30 trinh hr voi phan tuyp tir 2a den 2r. D61 vOi
kieu gen 6 co tat ca 83 trinh hr thu6c phiin tuyp 6a den 6w. Kieu gen 3 co 10 trinh tu veri phan tuyp 3a den 3k, kieu
gen 4 co 15 trinh to tir 4a den 4t, kieu gen 5 c6 hai trinh to va 1 trinh to GBV thuOc nhOm ngoai (outgroup).
Khito sat in silico ye 2 SNP rs12979860 vi rs8099917
Dji vai rs12979860
Bie'n the C/C chi4m tjt le cao nhat (40,99 %), bien the TIT chiem t' le thap nhat (13,01%). Ngueri da tang co
ty le C/C là cao nhat (42,45%), ngtriri da den thi ty' le C/T cao nhat (41,89%). Su phan bO cac bign thg theo dia
lY }thong co su. chenh lech clang kg: Bien the C/C luon chiem ty' le cao nhat va T/T luon chigm ty' le thgp nhfit.
Qua ket qua thong Ice kha nang dap Crng virus keo dai (SVR) cua tong bien the chung toi nhan thay hien the
C/C c6 SVR cao nhat (80%), bien the T/T co SVR thap nhat (12%).
Doi vai rs8099917

136


Ky yiu khoa hoc

Giai thuimg SV.NC1CH-Eureka lain thir 15 nam 2013

CO so khac biet giaa cac khu voc dia ly nhung nhin chung bien the T/T luon c6 tan so xuat hien cao nhit
(60%), bien thEG/G co ten s6 xuat hien it nhat (5%).Bien the T/T c6 SVR cao nhat (37%), G/T va G/G c6 dap irng
dieu tri thap han (20%).
Khio sat sir hoyt dfing cua moi khuech dyi Wing trinh tv chink SNP tren thirc nghiem
Qua trong qua trinh tham khao va danh gia he mai - mau d6 sir dung trong phuang phap real-time PCR chung
da chon ra &roc 2 cap mai toi tru nhat xac dinh 2 SNP quan tam. Trinh to mai sau de da duqc chung tei chinh sira
bang phan mem bioedit nham phis hop han yeti nghien ciru.

Buac clau kiem tra so hoot deng, de dac hieu va nhia de lai tai tru dm 2 cap mei ten thoc nghiem. Ket qua
cho thay ca 2 cap mei deu hoot dang tat, san phirn khuech dai co kith thuac bang vai kith thuot dy kien (71bp clei
voi rs12979860 va 118bp dej vai rs8099917). Khuech dai dac hieu Wing trinh to chira SNP quan tam va nhiet dcj lai
toi uu caa 2 cap mei Ian !trot la 52°C va 54°C.
Hinh B

Binh A

200 bp___„
118 by

Hinh . Ket qua cli0i di sari phAm PCR db tini Tin toi thictl cho clip ni6i RS860
(With A) va

inOi RS917 (hinh B). Thang chuin 100 by

RT-PCR, giii trinh ty. va phfin tich phi hg phfin tin
12 mau benh pham mau deu cho ket qua RT-PCR dot chat Itrang tat, du de gai giai trinh to (da lieu khOng
trinh bay). Ket qua giai trinh to dirge doc tren phan mem Chromas Pro 2.0.4, phan tich vai tong co Blast. Ket qua
truy xuat to tong cu BLAST cho thay trinh to cac mau san pham RT-PCR thuec clang cac kieu gen HCV (du lieu
khong trinh bay).
Trinh to NS5B tir 12 mau benh pham va cac trinh to tham chieu (khoang 340 nucleotide/ 1 trinh tu) yang
NS5B cua cac kieu gen 1, 2, 3, 4, 5 va 6 HCV va trinh tin thuec virus GBVC, HGV (NC_001710) doge dung cay
pha he theo ca 3 phuong phap Neighbor-joining (NJ), Maximum likelihood (ML) va Maximum parsimony (MP)
vOl gia tri bootstrap lap lai 1000 Ian. Ka qua cho thay co so phan.b6 hop ljr giira cac kieu gen va phan tujp trong dia
hinh hoc (topology) cha ca 3 cay phi he. Gia tri bootstrap deu the hien do tin cay cao (Hinh 1). Tinh hop nhat ye' so
phan be cha 6 kieu gen va cac phan tuyp deu dirge the hien tren ca 3 cay pha he cho ca nhOm trinh Or tham chieu Ian
12 trinh to giai &roc trong nghien ciru nay. Ket qua nay duor tai kiem tra bang each chung toi that hien Blast vai
12 trinh to not tren va sir dung ccr sa din lieu caa GenBank, di, day kh6ng phai la phucmg An hoan town tai iru. Tuy
nhien, ca hai ket qua deu the hien tinh phis hop (da lieu khong trinh bay).

137


Giai thitemg SV.NCKH-Eureka kin thir 15 num 2013
(A)

K.))yiu khoa hoc
(C)

(B)
_11

—0----=
r

_v.—ammo

-.1

•
911.-1
•

•

_kw.

-•-

-•-•-4-

•

Hinh 1. C5y pha he HCV du:crc clung theo 3 phuang phial): (A) Neighbor-joining, (B)
Maximum likelihood (C) Maximum parsimony
Tr ket qua nay, chimg toi xác dinh kieu gen va phan tuyp HCV tir 12 miu berth pham nhu sau: (1)
Trinh ti,r NS5B a 3 mill thuec kieu gen 1 (m5u X3601, X3601-6-9 thuec phan tuyp la, mau VA-1 thuec
phial tuyp lb). 4 mau thuec kieu gen 2 (4 mu X3602-7-9, 1317, X3602-6-9. X3602-14-8 thuec pilau tuyp
2m), 5 m5-11 duce xac dinh la kieu gen gen 6 (inau X3603 va VA-6 thuec phan tuyp 6a. mau X3602 va
X3604 dtrac xep vao pilau tuyp 6e, trinh tu X3604-15-8 duric xep vao pilau tuyp 6t).

138


1C)).014 khoa hoc

Giai thuzing SV.NCKH-Eureka ldn thti 15 ncim 2013

Bing 1. So sanh phiyang phap giai trinh ttr met phhn ming NS5B va Real-time RT PCR tren vung 5'UTR
KM qui phan tuyp HCV vai
phu'o'ng phip NS5B

Dinh ki6u gen bang phu'o'ng
phi', Real - time RT PCR (1
Kik' gen 1
Ki6u gen 2
Kitting dinh duvc ki6u gen
Kik, gen 1 hoic 6
Tong miu

1a lb
2 1

2m

6a 6e
2

6t
1

6

1

4
1
1
12

4
1
1

2

1

4

2

2

Tong mau

Trong qua trinh tien hanh thuc nghiem buErc au nay, chimg toi so sinh kat qua xac dinh kieu gen va phan tuyp
0 day vEri met phtrang phap khac sir dung be kit ctia ding ty Viet A dua tren kjr thuat real-time RT PCR dua tren
vimg 5'UTR. Ket qui so sanh nay nhu sau (Bang 1): phtrong phap real-time RT-PCR va phtrang phap nay cho kat
qua xac dinh kieu gen diing nhit a 7 mau (3 mau — kieu gen 1, 4 mau — kieu gen 2). Co 3 mau &rot xac dinh thuec
kieu gen I beri phtrong phap real-time RT-PCR nhtmg phuang phap giii trinh hr cho ket qua thuec kieu gen 6.
Ngoai ra, co 1 mau (VA-6) real-time RT-PCR khong xac dinh chinh xac kieu gen ma ghi nhan 1 hoac 6, thi giii
trinh to khAng dinh la kieu gen 6. Nhu vay, rO rang la giii trinh to to ra hieu qua hon: khong nhting tra IEri &rot kieu
gen ma con cho biet phan tuyp; ngoai ra, khong the gay nhim Ian gitra kieu gen 1 va 6 (va day ding la van de da
&roc cac ding bo tren the giEri ghi nhan, khi xac dinh kieu gen dua ten vimg trinh to 5'UTR).
0 Viet Nam da c6 nhieu ding trinh nghien cua va img dung cac k'y thuat sinh hoc phan tir (RT-PCR va giii
trinh tu) trong vies phat hien va xac dinh kieu gen HCV. Wing NS5B cung da ducm met so tat gia trong nu& khai
thac nham xac dinh kieu gen HCV. Tuy nghien ctin nay chi mai ducrc thus hien vEri so luting mau benh khong
nhieu nhtmg diem khac biet la chung toi di )(Ay dung met phtrong phap luan nghien ciru, trong do met ca sa dir lieu
cue be dang tin cay claw xac lap dung lam tham chieu trong ba cay pha he phan tir neu ten de xac dinh kieu gen
HCV. Chinh dieu nay da dua den kat luan chinh xac ve kieu gen va phan tuyp HCV trong 12 mau benh phim.
KET LUAN
Qua thuc hien nghien ciru nay, chung CM rig ra ducrc met se, kat luan nhtr sau: (I) Da xay dung ca s6 dit lieu
cue be va thiet lap quy trinh thuc nghiem bang phtrong phip giii trinh to met phan yang NS5B HCV va buErc au
da cho kat qua dinh kieu gen va phfin tuyp ctia cac dung HCV trong12 mau benh pham trong khi real-time RT-PCR
chi xac dinh dugc a mire kieu gen. (2) Du that nghiem chi mai hen hanh tren 12 mau nhung,chang toi cung ghi
nhan kieu gen 1, 2 vi 6 la 05 Wen trong nhiem HCV 6 Viet Nam. Nghien dm nay se duostiep tut trien khai vol
di. Mau Ion hon trong thEri gian tot
DOi voi 2 SNP rs12979860 va rs8099917 tren gen IL28B co kha nang tien doan kat qua dieu tri 6 benh nhan

viem gan sieu vi C. Su phan bo cUa cac 'Jet bien nay c6 str khic nhau gift cac eking tic, khu vac dia ly nhtmg nhin
chung 2 bien the C/C cua rs12979860 va Trr ctia rs8099917 luen co tan so xuAt hien va kha nang dap mg dieu tri
cao nhat.

TAI LIEU THAM KHAO
Baclig MO, Chan VF, Ramos JD, Gopez-Cervantes J, Natividad FF (2010) Correlation of the 5' untranslated
region (5'UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping. Int J Mol
Epidemiol Genet, 1(3): 236-244.
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Gidi thtrerng SV.NCKH-Eurika ldn thin 15 nean 2013

GENOTYPING HEPATITIS C VIRUS BASED ON SEQUENCEANALYSIS
OF THE NS5B REGION
Le Huyen Ai Thuy' *
'Faculty of Biotechnology, Ho Chi Minh city Open University
2Medic central HCM city
3 Viet A Technology -Corporation Company
SUMMARY
Hepatitis C virus (HCV) is blood-brone pathogen leading global liver diseases and possess significant threat to
public heatlh worldwide. Recent studies showed that different HCV genotypes are associated with pathogenicity,
infectivity and response to treatment regimens in special situations. The non-structural 5B (NS5B) region encodes
the RNA-dependent RNA polymerase, this is the variable sequence which is considered as the reference sequence
for indentifying both genotypes and subtypes of HCV.
The objective was to collect a source of information and data about genotypes and subtypes in Vietnam. The

experimental study develop a method to amplify and sequence, phylogenetic analyse base on NS5B region by using
HCV RNA isolated from the plasma samples of patients chronically infected with HCV.
For the 12 samples with NS5B sequencing, phylogenetic analysis showed that concordance of genotypes and
subtypes with a bootstrap > 90% for each cluster. Those were subtyped as genotype la in two samples, genotype 1 b
in one sample, genotype 61 in one sample, genotype 2m in four samples and genotype 6e in others.
Our resutls suggest that HCV NS5B region genotyping method has more advantaged and preferable than realtime PCR method for epidemilogical investigation of HCV. Thus, our study should be further tested with the large
sample sizes.
Keywords: Hepatitis C virus (HCV). NS5B region, sequencing, RT-PCR. phylogeny

*Author for corresspondence: Email:
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