Y HQC Vlf T NAM T H A N G 1 S6 2/2012
PHAT HIEN VIRUS ffUBiU/limm D|CH 01 THAI PHU BANG KY THUAT NESTED PCR
Vu Xuan NghiaS Pham OuTc M i n h \ Nguyen Quang Bac^
Oang Tien TrUdng\ Nguyen Thu Thuy^
TOM TAT
Djch sot Rubella trong thdi gian gan day co dau hieu bung phat trd lai. Bdi vay, chan doan nhanh va chinh xac mam benh se giiip cho cac bac si CO chi dinh dieu tri phii hdp doi vdi thai phu.
Trong nghien cifu nay, ky thuat Nested PCR da Ung dung de phat hien virus Rubella trong dich 6i, gian tiep chan doan nhiem virus tren thai nhi. BUdc dau Ung dung nested PCR tren 41 benh nhan nghien ciru CO bieu hien nhiem virus Rubella, phat hien 13 tru'dng hpp (32%) co RNA ciia Rubella trong die h di.
Vdi Uu diem nay, ky thuat nested PCR co tlu- dUdc Ung dung trong chan doan trUdc sinh.
Tdkhoa: Nested PCR, RNA, virus Rubella.
ABSTRACT
DETECTION OF RUBELLA VIRUS IN AMNIOTIC FRUIT FRAGNANT WOMEN BY NESTED PCR
In recent years, the outbreaks of Rubella fever have flared up again. Rapid and accurate diagnostic helps doctors to indicate the pathogen eariier in fragnent women. In this study, nested PCR technique was applied to detect rubella virus in amniotic fluid, indirect diagnosis of viral infection on the fetus. Initially, nested PCR technique was applied on 41study patients having symptoms of aibella virus infection, 13 3ses (32%) had RNA of Rubella in amniotic fluid. With this advantage, nested PCR technique can be applied in prenatal diagnosis.
Key words: Nested PCR, RNA, Rubella virus.
I.
OAT VAN OEVirus Rubella, thude ho Togaviridae, ed mdt bd gen ARN sdi ddn. Virus lay truyen qua dUdng hd hap, cU trii tai vdm hong va cac hach bach huyet. Benh nhan sau nhiem tU 5-7 ngay, virus lUu hanh trong mau va lay lan khap cd the. Dac biet virus Rubella cd the vUdt qua nhau thai, ISy nhiem cho thai nhi, lam ngirng sU phat trien ciia te bao hoac pha hiiy chiing.
Sdi OUc (tieng Anh: German measles hay rubella) la mdt benh truyen nhiem rat de lay do virus rubella gay ra, sau khi mae berih se de lai mdt mien dich sudt ddi. Virus Rubella ehi gay benh cho ngu'di va thudng la d tre em. Oien hinh CLia benh la xuat hien eae not ban dd (phat ban) ngoai da, ed the kem_^theo sdt va sUng hach bach huyet. Thai phu nhieni virus Rubella trong thdi gian mang thai cd the dan den cac bien chUng ' Hoc viin Quan y; ^ Binh viin Phu san trung Udng.
nghiem trpng nhu say thai, thai chet lUu va dj tat bam sinh. 05c biet trong 3 thang dau ciia thai ky, 90% thai phu se sinh ra em be bi hdi chUnq Rubella bam sinh nhU: diec, due thiiy tinh the, tat m i t nhd, t§ng nhan ap bam sinh, tat dau nhd, viem nao, mang nao, cham phat trien, gan to, lach to... Tre mac hdi chUng Rubella bam sinh se la ngudn truyen nhiem rat cao eho ngUdi tiep xiic, virus ed the dao thai nhiieu thang sau khi sinh [1], NhUng trieu chUng ciia benh sdt phat ban Rubella rat nhe nen rat khd dUdc phat hien, dae biet d tre em. Benh khdi phat sau 10 ngay den 15 ngay ke tU lue tiep xuc vdi ngudi benh.
Trieu chUng thdng thudng la met mdi, sdt nhe, nhiie dau, sUng hach keo dai tir 1 den 7 ngay.
NhUng ndt ban mau hdng min, bat dau tU mat va nhanh ehdng lan xudng than, sau dd xudng canh tay va ehan trudc trUde khi bien mat. Ban tdn tai tU 1 den 5 ngay, nhUng thudng gap nhat la 3 ngay. Trong khoang thdi gian tU 7 ngay trUdc phat ban va trong lue phat ban la thdi gian ma ngUdi benh cd kha nang lay benh cao nhat.
Oe cd du doan chi'nh xac va giam thieu dUdc nguy ed dinh chi thai vdi nhUng thai phu bi nhiem virus Rubella, ehimg tdi ap dung ky thuat nested PCR phat hien virus trong dich di ciiajja me mang thai, nham chan doan tinh trang nhilm virus Rubella ci\a thai nhi.
II. VAT LIEU VA PHUONG P H A P NGHIEN CCfU 2.1. Benh nhan: 41 thai phu mang thai tUtuan thU 18, kham tai benh vien Phu san Trung Udng va vien quan y 103 trong 2 thang tU thang 7 den thang 8 nam 2011, dUdc xac dinh nhiem virus RubeHabar\g cac xet nghiem huyet thanh hpc IgM va IgG.
2.2. Benh phaim: Djch di 2-5 ml dUdc choc hilt tU thai phu nhiem virus Rubella, bao quan -80°C den khi su' dung.
2.3. Mau chu'ng: ChUng dUdng virus Rubella la mau virus chuan d\Jdc xac dinh bang kit realtime PCR (Primerdesign-England) tir djch di thai phu nhiem virus Rubella.
Odi chUng la virus Chikungunla,y\rus ciing ho Togaviridae va cd kha nang nhiem tU me sang eon, dUdc lay tU ngan hang virus thupc Trung tam nghien cUu sinh y dUdc hpc-Hpc vien Quan y (Malaysia eung cap).
66
Y HOC VIET NAM THANG 1 - SO 2/2012 2.4. Ky thuat siT dung trong nghien cUu
2.4.1. Thiet ke moi
Mdi nested PCR dae hieu vdi virus Rubella dUde thiet ke bang phan mem CLC Genomics Workbench 4.0.3 (CLC bio, Oan Maeh) dUa tren dU lieu ed sd eiia Trung tam thdng tin edng nghe sinh hpe qude gia Hoa Ky NCBI (National Center for Biotechnology Information), trung tam dU lieu mam benh virus Hoa Ky VIPR (Virus Pathogens Resources) va trung tam du' lieu virus RNA (RNA Virus Database). Gene dich thudc gene ma hda protein mang E l . Mdi dUdc thiet ke Rul_A: 5'- AGAGRGTTCCGTWACGGTArTCGG -3'va Rul_B:
5'- AATRATAGCWGTGTCACYCGT -3'; Ru2_A: 5'- CGWTGYTACATTGGWACTCGARTGGG -3', Ru2_B:
5'- CTCCRCYCTAAV\rrGATAYCATA -3'.
San pham nested PCR cd dp Idn 280 bp (trinh tU tham chieu tU AY161356.1 Rubella virus isolate 4007-ITA91 El protein gene).
2.4.2. Tach chiet ARN: ARN eiia virus dddc taeh tU 150 pi djch di bang Qiagen ARN mini kit thed quy trinh ehuan eiia nha san xuat (Qiagen, OUc) va
0
eat giU d -80 C den khi sU dung.
Z4.3. Phan uhg khuech dai gen Nested PCR Kit Qiagen onestep (Qiagen-OUc) dUdc sU dung khuech dai gene dich. Thanh phan tham gia phan Ung va ehu trinh nhiet difdc tdi Uu hda.
Chu trinh nhiet difdc thifc hien: ehuyen ddi ARN thanh cADN tai 45°C trong 45 phut. PCR vdng 1:
95°C trong 2 phiit va 40 vdng eiia eae giai ddan:
duoi xoan d 94°C trong 30 giay, bam mdi 55°C trong 1 phut, keo dai 72°C trong 1 phut. Cudi cung la giai doan keo dai 72°C trong 10 phiit.
Phan Ung PCR vdng 2: 95°C trong 2 phiit va 40 vdng ciia cac giai doan: duoi xoan d 94°C trong 30 giay, bam mdi 57°C trong 1 phut, keo dai 72°C trong 1 phut. Cudi ciing la giai doan keo dai 72°C trong 10 phiit. Sau khi nhan len, san pham PCR chay tren agarose gel 1,2% d dien ap lOOV va chup tren he thdng may doc gel.
2.5. Thiet bj, hoa chat
2.5.1. Thietbn dung eu va thiet bj ehuyen dung eiia phdng Vi sinh y hpc va mam benh Trung tam nghien eUu Y DUdc hpc Hpe vien Quan y gdm: may luan nhiet tU ddng GeneAmp PCR system 9700 AB (Applied Biosystems, Hoa Ky), may ly tam lanh Mikro 22R (Hettieh, OUc), may soi va chup gel Dolphin Doe (Waltee, Hoa Ky), bd dien di.
2.5.2. Hoa chat: Cac hda ehat ehinh d\fdc sU dung la: dung dich dem HB (8x SSC; 0,5% SDS),
nUde khU ion, thaeh Agarose, SYBR-green de nhudm gel, thang DNA ehuan do hang Sigma cung cap.
III. KET QUA NGHIEN CUU
3.1. Do dac hieu ciia moi tren mau chdng dWdng va ddi chdng: Oe tdi Uu hda quy trinh, ky thuat nested PCR d\fdc ap dung phat hien ehUng dUdng. Ket qua eho thay, san pham PCR eiia ehUng dUdng Rubella chay tren agarose gel 1,2% vr^i dien thp 100v dat 280 bp.
300DP
Hinh 1: Op dac hieu eiia mdi Rubella in vitro.
1. Thang DNA Ikb; 2. Virus CHIK; 3. Chu'ng am;
4. Chu'ng du'dng virus Rubella.
Ket qua hinh 1 eho thay mdi nested PCR cd kha nang phat hien virus Rubella vdi dp dae hieu 100%, khdng ed phan Ung cheo vdi virus Chikungunya.
3.2. Phat hien RNA cua virus Rubella trong dich di: Djeh di eiia thai phu nhiem Rubella sau khi d\idc lay, ly tam, taeh ehiet ARN.
San pham dUde sU dung ehay RT-PCR vdi eae cap mdi nested. Ket qua cho thay, trong 41 mau dich di cua thai phu dUdc ehan doan nhiem virus Rubella, nested PCR phat hien 13 mau (32 %) ed RNA ciia virus Rubella.
300bp 28063
Hinh 2: Phat hien RNA ciia virus Rubella trong djch oi. l.thang DNA Ikb; 2. Chdng am;
3,4,5,6. Dich oi; 7. Chu'ng du'dng Ket qua hinh 2 eho thay, ky thuat nested PCR phat hien RNA eiia virus Rubella trong mau dich di thai phu (mau 4 va 6).
3.3. So sanh kit qua Nested PCR vdi chan doan huyet thanh hgc Ig.
Oe danh gia y nghia eiia phUdng phap Nested PCR, ket qua eiia eae mau ehay d\idc so sanh vdi chan doan huyet thanh hpe IgM va IgG (bang 1).
67
Y HOC VIET NAM T H A N G 1 s 6 2/2012 Bang 1 : So sanh ket qua xet nghiem huyet thanh hoe va Nested PCR
PhUdng j _ Huyet thanh hpc phap
Ket qua
IgM
41/41 (100%)
(i i
0 (0%)
IgG
41/41 (100%)
'1
0 (O'Vo)
Nested PCR
13/41 (32%)
i±L
28/41(68%)i±
Ket qua cho thay, phUdng phap nested PCR phat hien 13 (32%) thai phu trong dich di ed nhiem virus Rubella khi IgM va IgG dieu ( f ) .
IV. BAN LUAN
Hien nay, djch sdt phat ban biing phat ngay mdt phUc tap d nUdc ta, dien hinh la benh Ruttella, vi vay ddi hdi phai cd nhUng edng eu ehan doan phat hien nhanh va chinh xae eae mam benh gay ra. TU dd, cd nhUng bien phap thich hdp trong dU phdng va dieu trj [ 1 ] , [ 5 ] , [6],
Oe phat hien virus Rubella, mdt sd phUdng phap dUdc thuc hien thUdng quy d labo nhU:
phan lap virus, phat hien virus bang ky thuat mien dich dac hieu va phat hien virus d mUe dp gen bang eac ky thuat khuech dai chudi acid nucleic [ 2 ] , [ 3 ] , [ 4 | . Mac dii la phUdng phap mang lai tieu ehuan vang trong chain doan, nhUng phUdng phap phan lap virus cd nhieu nhUde diem nhU thdi gian keo dai (khoang 7 ngay), labo trang bj tdt va ddi hdi ky thuat vien cd trinh dp cao. Viec phan lap virus cung gap khd khan do nudi cay virus Rubella rat khd va ndng dp virus trong mau thap, nhat la d giai doan toan phat eua benh. Trong khi dd, phUdng phap huyet thanh hpc, phat hien IgM va IgG khang virus Rubella da ehUng td dUdc Uu the nhU thdi gian phat hien nhanh, de sU dung, khdng ddi hdi ky thuat vien cd trinh dp cao va cd the lam ngoai labo. Hdn nUa, phat hien eac Ig cd the dUdc tien hanh bang que thU nhanh hoac bang kit ELISA cd gan xhang the dac hieu.
PhUdng phap sinh hpc phan tU dUa tren phat hien trinh tU genome ciia virus nhU: RT-PCR (Reverse Transcription-PCR), Nested PCR, Realtime PCR. Oac diem ciia nhii'ng phUdng phap nay la cd the nhan hang ty lan mdt doan gene dac hieu ciia vi sinh vat cd trong benh pham vdi mdt khoang thdi gian ngan ma khdng can den qua trinh nudi cay. Trong khi dd, phUdng phap truyen thdng phai mat ft nhat tir 5 - 7 ngay.
Ky thuat Nested PCR dUa thUc chat la hai phan irng PCR d\idc thUc hien ndi tiep nhau. San pham ciia phan Ung PCR thir nhat se dUdc diing lam khudn cho phan Ung PCR thU hai, nhd dd ma dp nhay va dp dac hieu ciia phan Ung tang len.
Hien tai, cae ehan doan bang PCR deu cd thien hudng dUa tren ky thuat Nested PCR hoac Realtime-PCR.
Trong nghien cUu, ky thuat nested PCR dUa 68
tren cac c3p mdi da dUdc thiet ke cho thay tinh dae hieu eao trong phat hien RNA eiia virus
Rubella va khdng cd phan irng cheo vdi virus Chikungunya,
BUdc dau Ung dung tren 41 mau dich di ciia eae thai phu dUde chan doan n h i l m virus Rubella bSng phUdng phap huyet thanh hpc, ky thuat nested PCR da phat hien dUdc 13 mau (32%) nhiem virus Rubella. Oieu nay rat cd y nghia trong ehan doan tinh trang nhiem virus ciia thai nhi. Hien nay, gan n h u tat ca cac thai phu nhilm virus Rubella trong 3 thang dau ciia thai ky co IgM hoae IgG dUdng tinii se dUde khuyen dinh ehi thai nghen. NhUng vdi ket qua eiia phUdng phap Nested PCR trong nghien cUu nay, ti le dinh chi thai nghen se giam mac dii thai phu cd IgM va IgG dUdng tinh trong mau. Bdi vay, ky thuat nested PCR dUdc ap dung phat hien RNA ciia virus Rubella trong dich di eiia thai phu ed y nghia rat quan trpng, giiip cac bac sT cd chan doan ehinh xae va dUa ra eac giai phap kip thdi ddi vdi thai phu.
V. KET LUAN
Ky thuat Nested PCR phat hien virus Rubella trong djch di cho thay cd kna nang phat hien vat chat di truyen RNA ciia virus Rubella vdi dp dac hieu tuyet ddi tren in vitro.
BUdc dau Ung dung, ky thuat nested PCR phat hien dUdc 13 mau nhiem virus trong 41 mau (32%) dich di ciia thai phu da dUde chan doan nhiem virut Rubella bang phUdng phap huyet thanh hpe.
T A I LIEU T H A M KHAO
1. Best JM. (2007): Rubella. Semin Fetal Neonatal Med. 2007 Jun; 12(3): 182-92. Epub 2007 Mar 2.
2. Bosma TJ (1995): PCR for detection of rubella virus RNA in clinical samples. May 1995. 3 Clin /v//cro.33:1075-1079.
3. Mclver CD (2005): Development of multiplex PCRs for detection of com. 3 Gin Mien 43: 5102-5110.
4. Michel Segondy (1990): Detection of Rubella virus in amniotic fluid by electronic microscopy.
European 3ournal of Obstetrics & Gynecology and Reproductive Biology 31 (1990) 77-81.
5. Pandolfi E, Chiaradia G, Moncada M, Rava L, Tozzi AE (2009): Prevention of congenital rubella and congenital varicella in Europe. Euro Sun/elll; 14(9): 16-20.
6. Plotkin SA (2001): Rubella eradication. Vaccine.
2001 May 14;19(25-26):3311-9.