NGHIEN CUfU DjCH T I HQC PHAN TUT CAC CHUNG VIRUT SOI Lim HANH TRONG CAC VU DICH SOfI NAM 2006-2013 6 MIEN BAC VIET NAM
v u THI KIM LIEN; D 6 THj QUYNH NGA; TRAN THj HAI AU.
N G U Y I N TH! HONG NGOC*; TRI$U THI THANH V A N ; D 6 PHl/ONG LOAN; N G U Y I N THAI SON*
Vi&n V& sinh Dich te Trung wong
* Hpc vien Quan Y103
T 6 M TAT
Doan ADN 492 bp ddc hidu genom Nucleopmtein vims s&i cua 43 chOng vlmt s&i phdn ldp tmng cdc vu dich sdi tai Hd Ndi. Thanh Hod; Bic Giang. Hod Blnh;
Nghd ^"- Hd Giang; Lai Chdu, Ldo Cai... dwgc phdn tlch bing phwang phdp PCR-RFLP vd gidi trlnh ty Kit qud dd xdc dinh dwgc sw iwu hdnh cOa genotype HI virut s&i t^i mien Bde Vi&t Nam tmng giai doan 2006-2013. phu hap v&i kit qud phuong phdp gidi trlnh tw ADN Do vdy. phuong phdp PCR-RFLP cd thi dwgc sO- dyng trong cdc nghi&n ciru nhim xdc dmh nhanh ddc aiem dich te hoe phdn tO- cOa cdc chiing S&I dang Iwu hdnh.
Tip khod vimt s&i. RFLP; gidi trlnh tyADN SUMMARY
MOLERCULAR EPIDEMIOLOGY OF MEASLES IN THE NORTH OF VIETNAM
The nucleopmtein gen N of 46 confirmea measles isolates In Hanoi. Thanh Hoa, Bac giang, Hoa binh.
Nghe an, Ha giang, Lai chau, Lao eai between 2006- 2013 wem analyzed by PCR-RFLP and sequencing PCR-RFLP analysis mvealed the circulating of genotype HI in collected Isolates. The sequence of 660 ftp specific DNA of measles vims confirmed the don .iiant of genotype HI in Vietnam, 2006-2013.
Thus, PCR-RFLP method can be used for molecular epidemiology analysis of measles vivus.
Keywords Measles vims, restriction fragment length polymorphism; sequencing DNA.
DAT V A N Dt
Theo t i chde y t l t h i gidi (WHO), tinh trang t u vong do bdnh sdi da giam 78% vd vaccine dd cuu sing 4,3 tridu ngudi trong thdp ky qua Tuy nhidn theo thing kd cua WHO trung binh trdn the gidi mfli ngdy cfl khoing 380 tre em chet vl sdi hign vdn la qud Idn d i i vdi m i t bdnh hodn todn cd the phflng ngira dup-c blng vaccine [1]. Tii- ndm 1998, nghidn CU'U djch t l hpc phdn tir n h i m xde djnh ede genotype Chung virus sdi hoang dgi luu hdnh tgi edc viing dia ly khdc nhau dwgc WHO d i y mgnh, Mue tidu cua chiln lup-c ndy nhdm k i l m sodt, ngdn chdn sy ldy truyln cua virus sdi, t i l n tdi khing e h i bdnh sdi tren todn c i u D l dgt duge d i l u ndy, WHO kdu gpi sy
hop tdc eua t i t ca cdc q u i c gia trong vide hodn thidn ban d i phdn b i genotype cua todn bfl chung virus sdi hidn dang luu hdnh tgi cdc viing dia ly dan eu trdn khip edc Chdu luc [2]
Tai Viet Nam, cde vu djch sdi d mpi lua tuii. xay ra tai nhieu dia phuong trong nhung ndm gan ddy Id v i n d l noi com trong ngdnh y te dy phflng, Chinh vi vay vide xdc dmh cdc genotype sdi hidn dang luu hanh giiJp cho vide hodn chinh ban do phdn bo chung virus sdi tgi cdc viing lanh t h i quic gia Id v i n d l c l n thilt eho cdng tde giam sat dich td hoc
VAT LIEU VA PHU'ONG PHAP NGHIEN CU'U I.Vatli^u
- Chung vacxin sdi: chung virut sdi duoc phdn Idp t u ba loai vacxin s i n g giam ddc lyc gom:
W6675/Rouvax Aventis Pasteur (Phap); AIK-C Vero vd MMR (My) Cdc chung ndy duoc su dung nhu chung chuin trong nghidn cuu genotyp virut sdi
Chung d i i chung- ehiing vinjs sdi MVi/Hanam.VNM/6,09/9 HI (Genbank' JF824649) phdn Idp tip djch sdi tai Hd Nam thang 9/2009, dup-c su dung lam chimg d i i chung d l so sdnh vd phdn tlch vdi cdc chung hoang dai trong nghien ciru,
- Chimg vinit sdi hoang dd. 46 chung vmjs sdi thu dup'c tir mdt s l vu dich do Phflng virut hd hip. Khoa Virut, Vidn Vd smh Dich t l Trung uong phdn Idp tu mdu dich h i u hpng cua bdnh nhan cfl bilu hien Idm sdng bdnh sdi tu 2006 den 2013 tai mpt so tinh mien B i c Viet Nam- 6 chung ndm 2006 (Thdi Nguydn, Hai Duong, Thdi Binh, Didn Bidn), 15 ehiing ndm 2008 (Nmh Binh, Hd Nfli, Thanh Hod, Hai Duong); 13 chiing ndm 2009 (Nmh Binh, Hai Duong, Bde Giang, Hd Nfli, Lai Chdu, Hod Binh); 3 chung ndm 2010 (Nghd An, Hd Giang) vd 8 chimg 2013 (Lai Chdu, Ldo Cai) Cdc mdu dich hiu hpng ndy duoc phdn Idp trdn dflng t l bdo Vero/SLAM, n l u xuat hidn su huy hoai t l bdo, diing phuong phdp RT-PCR khdng dinh virus sdi,
2. Phuang phdp nghien cii-u
2.1. Phuong phdp tdch c h i l t ARN cua cdc chung virus sdi
RNA dup'c tdch chiet t u nudc n l i t l bdo gdy nhidm virus sdi theo hudng ddn cua bfl smh phim QIAamp Viral RNA Mini Kit (Cat No 52904_
V HOC THirr HANH ( 8 8 6 ) - S O 11/2013
QIAGEN)
2.2. Phuong phdp RT - PCR vd Nested - PCR Genom vinjs sdi deu Id mpt ehufli RNA don. Phan ung RT-PCR g i m hai giai doan chinh Id phidn md RNA vinjs thdnh cDNA nhd enzym phidn md ngup-c vd t i n g hg-p DNA dudi tdc dung cua DNA polymerase su- dyng bg sinh pham Tag RT - PCR Kit (New Englangd BioLab) Mdt s i trinh t y c$p m i l ddc hidu: Cdp m i i eho quy trinh PCR: MN5, 5'- GCCATGGGAGTAGGAGTGGAAC - 3' (1113-1134 trdn GenBank NC_001498,1); MN6:5'- CTGGCGGCTGTGTGGACCTG - 3' (1773-1754 trdn GenBank NC_001498.1),
Cdp m i i cho quy trinh Nested - PCR, N f l , 5'- GATGGTAAGGAGGTCAGCTGG 3'; Nr7: 5' TCGGCCTCTCGCACCTA - 3'
2.3. Phuang phdp RFLP
Nhdm tdc gia t i l p tyc nghidn cuu k l thii-a vd phdt triln dya trdn k i t qua thu duge eua de tdi nghien eiru c i p bfl "Nghien euu ddc diem di truyen mflt so chung sdi dang luu hdnh d Vidt nam bdng phuong phdp RFLP", cde Chung virut sdi thu thap tir ndm 2002- 2005 duoe thye hien bdi cac edn bfl khoa hoc phflng Smh hoe phdn tii-, Khoa Midn dich vd Sinh hpc phdn tu-, Vidn Vd sinh dich td Trung uong [3].
2.4. Phuong phdp gial trinh t y ADN Trong nghidn euu ndy, ehiing tdi xde dinh trinh t y ADN theo phuong phdp enzyme hpc cua Sanger thflng qua viee su dung edc dideoxynucleotid,
San phim PCR cd dfl ddi 660 bp dugc khuech dai trdn genom virut sdi, s u dyng edp m i l MN5 vd MN6. Sau dfl san p h i m PCR duoc tinh sach b i n g bfl smh phim QIAquick PCR purification Kit (Cat. No.
28104_ QIAGEN) vd dwgc sir dung Idm khudn d l giai trinh t u ADN.
KtT QUA VA BAN LUAN
1. RT-PCR k h u l c h dai doan gen N ddc hieu virut sdi
ARN sdi dup-c phldn ma b i n g Reverse transcriptase vd khuleh dai b l n g Taq polymerase.
S i n phim quy trinh PCR co do dai Id 660 bp. quy trinh Nested-PCR su- dung ADN khufln la s i n p h i m ciia quy trinh PCR vd san pham nay cfl dfl ddi Id 492 bp, San pham ciia quy trinh Nest-PCR khdng djnh ring vach 660 bp d i u tidn dung la mflt dogn ciia virus sdi dug-c nhan ldn (Kit q u i khdng duoc dua ra).
2. Md hinh djch t l hoe phan ttr cua cdc chung virut s d i su* dung p h u a n g phdp RFLP
Nhdm nghidn cuu su dyng enzym gidi hgn Mbol c h i tao Id san p h i m cua de tdi KHCN c i p thanh p h i (md s i : 01C-08/15-2012-2), dup-c lya chgn cho phdn tich genotype cua 46 chiing virut sdi phdn lap tu- cdc vy dich trong ndm 2006-2013 tai m i t s i tinh mien B i c Vigt Nam.
Theo tinh todn ly thuylt khi Mbol c i t ehung ehuln A_Edmonston-wt/54 (Genbank' U01987) cho cdc vach DNA vdi kich thudc 183, 142, 115 vd 52 bp;
Chung H1_Hunan,CHN/93/7 (Genbank: AF045212) eho vgeh; 167, 136, 106, 55 vd 28 bp, ehiing dli chirng H 1 _ Genbank; JF824649 cho vgch: 242, 155, 55. 29 v d i 1 6p.
K i t q u i dign di thyc nghidm cua chOng chuin.
Chung d i i chO-ng vd mflt s i chung virus sdi hoang dai phdn ldp tai m i t s i tinh M i l n Bde Vidt Nam trong giai doan 2006 - 2013 khi cdt vdi enzyme Mbo\ eho thiy:
chung vacxin cd hinh vach tuong t u chung chuin A_Edmonston-wt/54, chung virut sdi hoang dai cd cde hinh vach g i l n g nhau vd tuong t y vdi chung dli Chung H1_Genbank; JF824649 (MVi/Hanam.VNM/6 09/9 H I ) (Hinh 1). nhung kich thude cde vach ndy c i sy thay d i i so vdi chung chuin nhdm H I Id Hunan.CHN/93/7 (theo tinh todn ly thuyet), Tir d i cd t h i nhdn t h i y cd s y thay d i i v l vi tri nhan biet eiia edc enzyme ndy trdn trinh ty gen giua cdc chOng Vidt Nam so vdi chiing chuin Hu nan. CH N/93/7 H I (AF045212), xdc dinh dugc sy sai khae trong trinh t y giu-a cde ehiing Vidt Nam so vdi Chung ehuln.
Hinh 1 Ket qua RFLP chung virus sdi clt vdi enzym Mbol M Thang ADN chuin PBR-Mspl; 1; 3 Chung vacane, 2 Chiing virut sdi nhOm H1_Genbank-JF8246491, 4-17 cic
chiing virus sdi Thanh H6a. Nmh Binh, Hd, NOi, Bic Giang Khi phdn tlch chiing H1_Genbank, JF824649 vd cdc Chung virus phdn Idp tgi mdt s i tlnh miln Bic cho t h i y s y g i l n g nhau v l kieh thude vd s l luong cdc dogn DNA gidi han, do dd cho phdp nhdn dmh 46 Chiing virus hoang dai tii- ndm 2006 d i n 2013 trong nghidn cuu d i u t h u i c genotype H I , k i t q u i phii hpp vdi nghidn cuu trudc ddy [5]
Xdy dyng cdy p h i hd phdt sinh chung logi eiia kll qua phdn tich RFLP, 46 chung vinjt sdi bing phln m i m BioNumerics Fingerpnnt fypes_version 6.0 (phin m i m tin hpc b i n q u y l n cua Hdng Applied Maths _ B i ) (Hinh 2)
Y HOC THirC HANH (886) - S6 11/2013
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,L Hinh 2. Cay p h i hd phat smh chiing loai virut sdi blng phuong phap RFLP 3. M i hinh djch t l hpc phan tip ciia cdc chung virut sdi s u dung phuong phdp giai trinh t y ADN
Hinh 3: Cdy phdt sinh chung logi vinjs sdi miln Bde Vidt Nam giai dogn 2006-2013
Y HOC THUC HANH (886) - S 6 11/2013
Trong nghien cuu ndy, ehiing toi t i l n hdnh gidi trinh t y 44 tren t i n g s i 50 ehung vinjs sdi. Trong s l 44 chiing giai trinh t y g i m ed: 3 chung vaccine, chiing d i i chung H1_Genbank JF824649 vd 40 chiing vims hoang dai thu thap tgi Vidt Nam Cdc Chung giai trinh t y cfl s i thu- t u t u 1 - 44 trong danh sdeh chimg nghidn cdu.
Xdy dyng cdy p h i hd phdt sinh chung loai vinjt sdi luu hdnh tai Mien B i c Vidt Nam ndm 2006-2013. dya trdn k i t qua giai trinh t y ciia 44 ehung vinjt sdi bdng phan mem BioNumerics Sequence types vd BioNumerics Tme and Netwond infemnce (version 6.0 _ Applied Maths, BT). K i t qua cho thay todn b i 44 trinh t y phan tich d i u dugc phdn nhdnh genotyp H I , tuong t y vdi Chung d i i ehung MVi/Hanam,VNM/6.09/9 H I {Hinh 3) K i t q u i glai trinh t u ADN, phu hgp vdi k i t qua djch td hgc phan tir cdc chung virus sdi blng phuong phap RFLP. Do vdy, phuong phdp PCR- RFLP cd t h i dugc SLP dung trong cdc nghidn cuu nhim xdc djnh nhanh ddc diem dich td hpc phdn tii- ciia cac chiing sdi luu hdnh tai Vidt nam
K^T LUAN
- Xay dung thdnh eflng cay pha he phdt smh chiing loai virut sdi, m i i quan he di truyln vdi ehung chuan va cae chiing virut hoang dai khae nhau, s u dung phan mem BioNumerics Sequence types vd BioNumerics Tree and Netword Infemnce (version 6.0_Applied Maths, Bi)
- Xde dinh dugc genotype cua 46 ehung vinjs sdi luu hdnh trong mflt s i vu dich sdi tai Mien B i c Vidt Nam. giai doan ndm 2006-2013 thudc nhdm H I , si>
dyng phuong phdp -PCR-RFLP. Ket qua ndy dugc khdng djnh lai vd hodn todn phO hgp vdi k i t qua giai trinh t u ADN.
TAI LIEU THAM K H A O
1. Worid Health Organization (2012) 'Global measles and mbella stmtegic plan: 2012-2020"
2 Worid Health Organization (1998) "Weekly epidemiological record" No 35, 28 August 1998, 73.
pp 265-272,
3 Le Thi Kim T u y l n vd CS (2005) "Nghidn cii-u ddc d i l m di truyln mdt s i chung sdi dang luu hdnh d Vidt Nam bdng phuang phdp RFLP (Da dang d i ddi doan cat gidi han)", D l tdi c i p bg nam Y t l , nghidm thu 2005,
4. T r i n Nhu Duong, Nguydn Thi Thu Y i n , Phgm Quang Thai. Nguyin Thi Thu Huong "Mit s i ddc diem dich sdi tai m i l n B i c Viet Nam, 2006 - 2008°
Tap chi Y hoc d u phflng. Tap XXI, s i 5 (123) 2011, tr 37-45
5 _ Nguyen Hanh Phue, Nguydn Hiln Thanh, Nguyen Thj Thu Thuy vd CS (2009) "Genotyp HI vims sai Iwu hdnh tmng cdc vy dich s&l ndm 2006 - 2008 a mien Bic Vidt Nam", Tap chi y hoe dy phflng 2009. so 5(104), tr 5 0 - 5 6
TO! UU HOA DIEU KIEN SAN XUAT M A U BENH PHAM GIA DjNH MU, DOTM CHl)A VI KHUAN THUANG GAP PHUC VU KIEM TRA CHAT LimNG XET NGHIEM VI SINH LAM SANG
T R A N HUTU T A M , V 6 NGOC NGUYEN, TRUONG Q U A N THUY, NGUYEN TH! THANH T A M , N G U Y I N HlJfU L I ,
NGUYEN TH! THANH HOA, LE HIEU N G H T A . Trung tdm Kiim chuan X&t nghidm TP.HCM
TOM T A T
M6t trong nhung cdng cy di ddnh gid do tin cdy cua x&t nghiem vi sinh Idm sdng Id ngoai kiem tm chit Iwang (EQAs), de tridn khai dwoc hoat ddng ngo^i kiim tra chat Iwgng. dn phai cd cdc miu kiim chuin.
Dya tr&n kit qud khdo sdt cdc mdu benh phim vd vi khuan thw&ng gdp tai cdc phdng xdt nghigm vi smh ldm sang [3]. nhdm nghidn ciru dd xdy dyng quy trinh sdn xuit miu bdnh phim gid dinh mO, dam chira vi khuin gdy bdnh thw&ng gdp vd vi khuin thw&ng tni, tidn hdnh cdc thi nghidm sdng Igc vd tdi wu hda theo
ma tran Plackett - Burman, phwang phdp dw&ng doc nhit, phwang phdp hdm ddp ung be mdt - du true cd tdm (RSM - CCD) Kit qud da xdc dinh dugc Oiiu ki$n sdn xuit vd tim m dwgc cdng thirc tdi wu vdi cdc thdnh phin chi tidt cd thd dp dung sdn xuit hai miu b&nh phim gid dnh chdy vi khuin gdy bdnh vd vi khuan thw&ng trij gdm miu mu (S.aumus, P aemgmosa), miu d&m (Acinetobacter baumannii, P aemginosa, vi khuin thw&ng tru Stmptococcus mitis).
Tif khda: ngogi kiim, mO, d&m. Plackett - Burnian, RSM - CCD.
Y HOC THUC HANH (886) - sO 11/2013