PHAT HIEN VAT LIEU DI TRUYEN VIRUS CHIKUNGUNYA TIT BENH NHAN SOT XUAT HUYET BANG KY THUAT RT-PCR
Vu Xuan NghTa, Nguyen Linh Toan, Nguyen Trong Vien, Hoang Van Luong Hoc vien Quan Y, Hd noi
TOM TAT
Gan day virus Chikungunya (CHIKV) la mpt nguyen nhan gay ra dich sot xuat huyet (SXH) a cac nude Odng Nam A va Chau Phi Mac du, xet nghiem huyet thanh hpc cd the su dung de chan doan, nhung viec phat hien nhanh vat lieu di truyen ciia CHIKV trong mau benh pham van can duoc phat trien, hoan thien Trong nghien cuu nay, chung tdi md ta ky thuat RT-PCR true tiep (one step) khuech dai gen E1 ciia virus de phat hien tinh trang nhilm CHIKV tir mlu benh phim lam sang. Blng ky thuat nay da phat hien 4/50 (8
%) benh nhan sdt xuat huyet duong tinh vdi cap mdi dac hieu CHIKV. Phan tich, so sanh trinh tu vung gen E1 ciia mot mlu ducng tinh, xac dinh dp tuong ddng nucleotide so vdi chiing CHIKV S27 la 93 %.
Tir khoa: Sdt xult huyet, Chikungunya. DENV
I. DAT VAN DE
Virus Chikungunya (CHIKV) thuoc chi Alphovirus; hp Togaviridae [1], CHIKV giiy cac trieu chung lam sang nhu dau khdp, sdt va xuat huyet. day la nhung trieu chung lam sang ciing thudng gap tren benh nhan bi nhiem vi- rus O'ny ong-ny ong. virus Ross River, virus Barmah Forest va dac biet 0 cac nude Ddng Nam Chau A vii chau Phi. gay benh canh lam sang gidng vdi benh canh ciia Dengue xuat huyet [2], [3]. Vat lieu di truyen cua CHIKV cd cau triic RNA spi don, duong, cd kich thudc khoang 12.000 nucleotide |4]. CHIKV lan dau tien dupe phat hien d Tanzania (Chau Phi), su lan rdng ciia virus nay da dupe ghi nhan a nhieu nude va cac khu vuc tren the gidi. Gan day. CHIKV gay ra cac vu dich Idn d An Dp, Singapore, Thai Lan, Indonesia [5], [6] va dac
biet d Italia, mpt nude thupc Chau Au cd khi hau on hda [7]. Nhu vay. rat cd the CHIKV ciing la mpt trong nhirng nguyen nhan gay sdt xuat huyet d Viet Nam, nen trong nghien ciru nay chiing tdi sir dung ky thuat RT-PCR de phat hien vat lieu di truyen ciia CHIKV d benh nhan sdt xuat huy et
II. PHUONG PHAP NGHIEN CUU 2.1. Vat lieu
Man benh pham: Huyet thanh ciia 50 benh nhan cd chan doan lam sang sdt xuat huyet d giai doan sdm trong vu dich sdt xuat huyet nam 2009, dieu trj tai Berth vien Quan y
103 va Tram xa xa Ta Thanh Oai, Thanh Tri.
Hit Ndi.
Chiing duong (plasmid idi id hop) Chung duong cua CHIKV la mpt plas- Tac gia: Vii Xuan NghTa
Dia chi: Hpc Vien Quan Y. sd 114-Ha Ddng-Ha Npi Dien thoai: 0972925868
Email: nghia69'5;Gmail.com
IS2 Tap chi Y hpc du phdng, Tap XX. so 6(114)
mid bao gom toan bo doan gen co nguon gdc tir chung CHIKV S27. ma hoa cho protein mang El duoc tach dong (cloning) vao vec- tor pGOV4n (plasmid nhan duoc tir Malaysia theo chuong trinh hop tac nghien ciru).
Chung duong ciia DENV duoc thiet ke che tao tai Trung tam Y Sinh Duoc hoc Hoc vien Quan y sir dung san pham RT-PCR la mpt doan gen ciia DliNV dupe tach dimg trong v ector pGEMT.
Thong tin vd truth tit cup mdi.
Cap moi phat hien CHIKV dupe thiet ke bat cap dac hieu tren gen El ciia CHIKV chung S27 (AE490259) la E1F: 5'-ACCGG- CGTCTACCC-ATTTATGTG-3' va E1R: 5 - AGGGCGGGTAGTCCATG I TG-3' (33 I bp).
cap moi dupe thiet ke dua tren cac cong bo trudc day vii co chinh sua.
Cap moi ciia DE.NV la DI 5"-TCAA- TATGC •["GAAACGCGC-3" vii D2 5"-TG- CACCAACAGTCAATG 1-3" (5 1 1 bp) theo trinh tu nuclcotid ciia DENV type II da cong b6 |8|.
Ciic trang thiet bi va vat lieu can thiet dc thuc hien ky thuat RT-PCR. Cloning va Se- quencing.
2.2. Phuong phap:
Thdi gian lav tndu: Man mau duoc lav trong giai doan cap ciia benh. lay mau tinh mach vo triing. chiing dong. tach lay huyet tuong luu gii; a - 80"C cho den khi thuc hien
\ct nghiem.
'Tieu chndn ca benh: Benh nhan nhap vien vii co so v te voi bieu hien sot dot ngpt 38 - 39 ( . niem mac mat do. ket mac xung liny ct. not xuiu huyet tir nhien dudi da, nhieu o cang lay va diii. nhieu benh nhan cd dau co.
khdp. cd the cd gan to 2 - 3 cm dudi bp sudn.
mat dp mem.
AT thuat tach chich ARiX. ARN ciia virus dupe tach va tinh sach tu 150pl huyet tuong. su dung bp sinh pham ciia hang Qiagen Germany (Qiagen RNA Bipod mini kit) theo qui trinh cua nha san xuat. ARN thu dupe sir dung ngay clip phan irng RT-PCR hoac cat giir d -80"C den khi sir dung.
Quy trinh phan irng RT-PCR. Phan irng RT-PCR dupe thuc hien theo hudng dan ciia nha san xuat v di mpt sd thay dpi de tdi uu hda dieu kien phan irng. Thanh phan tham gia phan ling RT-PCR ban gdm cac sinh pham hoa chat can thiet nhu: 5 x Qiagen One step RT-PCR buffer x 5pl, dNTP 1 OmM/pl x 0.4pl. cap mdi phat hien CHIKV va DENV lOpmol/pl x Ipi (mdi primer), enzyme Taq-poly merasc x 2.5U.
ARN ciia virus va nuoc khu" ion khdng cd ARN vira dii 50pl. Chu trinh nhiet dupe thuc hien theo 45°C trong 45 phiit cho chuyen ddi tir ARN sang cDNA. Tiep den la 95 "C trong 2 phiit va 40 v dng ciia cac giai doan: dudi xoan d 94 C trong 30 s, bat cap mdi 55 °C trong I phiit, keo dai 72 °C trong 1 phut. Cudi ciing la giai doan hoan chinh san phiim 72 °C trong 10 min. Sau khi nhan len, san pham PCR ciia CHIKV va DE.NV dupe chay tren gel agarpse 1,2 % hieu dien the d 100 V va dupe chup tren he thong may dpc gel.
HI. KET QUA
3.1. Chuan hda phan ling RT-PCR.
De tdi uu hda dieu kien phan irng RT- PCR chiing tdi thuc hien phan irng tren plasmid tai to hpp mang gen El ciia CHIKV (chirng duong) sir dung cac cap mdi dac hieu tirong irng E1E-E1R cho CHIKV va D1-D2 cho DENV. Ket qua cho thay san pham PCR ciia CHIKV vii DENV chay tren gel agarose 1,2 % v di I OOv dat 331 bp va 5 1 1 bp rieng biet.
Tap chi Y hpc du phdng. Tap XX. so 6(114) 183
(A) (B) M P- P+
331 bp-
Hinh 1. Toi LFU hoa phan irng RT-PCR phat hien CHIKV (A) M: Thang chuan DNA 50bp;
P+: chu ng duong. cot 1: chung am; DENV (B) M: Thang chuan DNA 50bp; P+: chung dirong.
P-: chirng am
3.2. Phat hien vat lieu di truyen CHIKV trong mau benh nhan bang ky thuat RT-PCR.
Sau khi chuan hoa, ky thuat RT-PCR duoc sir dung phat hien vat lieu di truyen cua CHIKV va DENV trong mau benh nhan. Ket qua: trong 50 mau benh pham co 4 mau duong tinh la mau so 3, 6, 9 va 10 cho san pham PCR co kich thuoc la 331 bp tuang duong vdi kich thudc chung duong ciia plasmid.
Cac mau duong tinh vdi cap mdi dac hieu CHIKV duoc kiem tra lai vdi phan dug RT-PCR su dung cap mdi D1-D2 dac hieu DENV de phat hien DENV. Ket qua cho thay trong sd 4 mau duong tinh vdi CHIKV khdng cd mau nao duong tinh vdi cap mdi dac hieu DENV (Hinh 3). Trong khi dd, a nhdm chung am (n=10) khdng ed mau nao duong tinh vdi cap mdi dac hieu CHIKV va DENV.
511bp
Hinh 3. Ky thuat RT-PCR phat hien DENV trong cac mlu du ong tinh vdi CHIKV. Cot 1-4: am
tinh VOM DENV; P+:chtVng duong DEN; M:
Thang chuan DNA 50bp
Hinh 2. Ky thuat RT-PCR phat hien som CHIKV- RNA trong mau benh nhan. M: thang chuan DNA 50bp; Cot tir 1-10: Mau cua benh nhan; cot
11: chirng dirong.
IS4 Tap chi Y hpc du phdng, Tap XX. so 6 (I 141
500bp 250bp
Hinh 4. (A) Phan irng RT-PCR phat hien vat lieu di truyen CHIKV trong mau benh nhan.
(M). Thang chuan DNA (50 bp); (1) chung am: (2) mau duong tinh tCr benh nhan; (3) chirng duong CHIKV. (B) Phan irng RT-PCR dung cap moi dac hieu DENV (D1 ,D2): M, thang chuan DNA 50bp;
(1) mau benh nhan (am tinh); (2), chirng dirong DENV.
Trong so 4 mau duong tinh voi cap moi dac hieu CHIKV viing gen El duoc phat hien trong vu djch sot xuat huyet 2009. su dung nipt mau duong tinh de giai trinh tir gen. day la man duong tinh dupe phat hien tir mpt benh nhan nu. 28 tuoi den Tram y tc xa voi nhung bieu hien dien hinh ciia nhCrnu trieu chirng di
nhiem CI IIKV nhu sot cao dpt ngpt, dau diiii.
moi co. dau cac khdp ngay thii' 4 cua benh, tien su benh nhiin hoan toan khoe manh (trong thdi gian hai tuan trudc khi bj phiit benh, ngudi benh 6 tai xa Pa Thanh Oai, huyen Thanh tri.
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Hinh 5. So sanh trinh tir nucleotide cua mot phan viing gen E1 CHIKV phat hien 6> benh nhan (CHIKV-Vietnam) vm trinh tir cua mot so chung CHIKV tham chieu trong ngan hang gen voi so
dang ky AF359024, AF490259 va FJ445510.
Tap chi Y hpc dir phdng. Tap XX, so 6 (1 14) 185
San pham PCR tir mau duong tinh nay duoc tinh sach va lai ghep vao vector pGEMT (lao dong), xac dinh trinh tir acid nucleic ciia mau duong tinh. So sanh trinh tu vung gen El cua CHIKV phat hien tir benh nhan Viet Nam voi trinh tir viing gen El ciia mot so chiing CHIKV da dang ky trong ngan hang gen. cho thay su tuong dong ve nucleotide voi CHIKV co nguon gdc tir Chau Phi (chiing CHIKV S27) la khoang 93 %.
IV BAN Ll AN
Su thay doi nhanh chong ciia khi hau tren toan can dan toi canh bao ve cac vu djch do Arbovirus gay ra. Hien nay. CHIKV va DENV duoc canh bao la nhung Arbovirus quan trong nhat gay sot xuat huyet d nguoi.
CHIKV va DENV lay nhiem do ciing mot loai vector nen chimg luu hanh d nhirng khu vuc giong nhau, co nhiing bieu hien trieu chimg lam siuig kha giong nhau. Do vay, co gia thiet cho rang nhieu truong hop mac CHIKV khong duoc phat hien trong nhirng vu sot xuat huvct gan day.
I lien nay. tren the gioi va trong nuoc sii' dung nhieu ky thuat phan tii de phat hien cac (ac nhan sinh hoc. Trong do, co nhung ky thuat phat hien nhirng san pham true tiep hoac gian tiep cua tac nhan gay benh. Ung dung ky thuat RT-PCR co the xac djnh CHIKV va DENV trong mau benh pham lam sang bang nhirng ciip moi dac hieu ciia virus. Ky thuat RT-PCR true tiep (one step - mot giai doan) cho phep thuc hien dupe nhanh, thuan Ipi va giam thieu tdi da kha nang lay nhiem sp vdi ky thuat RT- PCR gian tiep (two steps - hai giai doan) |9], Su dung RT-PCR da phat hien dupe 1 50 (8 °o) mau mau benh nhan duong tinh
\ di cap mdi dac hieu CHIKV viing gen E. I. la
biing chiing cho thay sdt xuat huyet hien nay d nude ta khdng don thuan chi do DENV ma cdn do ca CHIKV. Day la dido can dupe quan tam nghien ciru mang tinh chat tdng the ca ve lam sang, virus hpc va djch td hpc. Cho den nay.
chua cd mpt cdng bd nao ve viec phat hien vat lieu di truyen ciia CHIKV a benh nhan ngudi Viet Nam. Mac dii. tai lieu Irudc day chp thay sir ton tai khang the khang CHIKV tren nhirng linh My tham gia trong chien tranh Viet Nam.
Cac mau phat hien duong tinh vdi cap mdi dac hieu CHIKV se dupe luu giir hy vong cd the phan lap dupe CHIKV trong nghien ciru tiep theo tir nhirng mau dii xiic djnh duong tinh niiy
iuy CHIKV dupe phat hien dau tien d Chau Phi 1952, nhirng rat nhanh chdng gay cac vu sdt xuat huy et Idn d cac khu vuc Chau Phi va Chau My . Gan day. cluing gay ra nhung vu djch Idn d Chau A vii dac biet la cac nude Ddng Nam A nhu: Singapore, Thai Lan, Indo- nesia vii An Dp.
Qua ket qua nay cho thay. cac vii djch sdt xuat huyet d Viet Nam. ngoiii nguyen nhan do DENV, cdn mpt virus khac cd the cung ddng vai trd gay benh do la Chikungunya.
Vdi ket qua ban dau nay cho thay nhung vu djch sol xmit huyet d nude ta khdng chi don thuan do DENV giiy ra ma sir thay ddi vc tinh chat, cudng dp ciia djch cd the phan nao cd phan ddng gdp ciia CHIKV. Chinh vi vay, can cd nhirng nghien ciru co- ban va toan dien ve CHIKV o nude ta.
V. KET IT AN
Ky thuat RT-PCR cd the sir dung de phat hien nhanh vat lieu di truyen ciia CHIKV trong mau benh nhan sdt xuat huyet vdi ty le da phat hien duong tinh la 4/50 (8 %) bang cap mdi dac hieu CHIKV viing gen li I khang djnh cd sir luu hanh cua CT 1IKV d nude ta.
Tap chi Y hpc du phdng. Tap XX. so 6(114)
TAI LIEU THAM KHAO
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gue: a case of mistaken identity? J Hist Med Allied Sci 26: 243 262.
3. Myers R. M.. Carey D. E. (1967). Concur- rent isolation from patient of two arbovi- ruses, chikungunya and dengue type 2.
Science.l57:1307 - 1308.
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Virol.88(Pt 9):2363-77.
5. Leroy E. M., Nkoghc D. (2009) Concur- rent Chikungunya and Dengue Virus infec- tions during simultaneous outbreaks, Ga- bon, 2007. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No.
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an epidemic arbovirosis. Lancet Infect
Dis.;7:319 327.
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(2007) Infection with chikungunya virus in Italy: an outbreak in a temperate region.
Lancet:370:1840 - 1 846. Lanciotti RS. Cal- isher CH, Gubler DJ, Chang GJ, Vorndam AV (1992) Rapid detection and typing of dengue viruses from clinical samples by us- ing reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30: 545-551.
8. De Paula S. O.. de Melo Lima C Torres M. P., Pereira M. R., Lopes da Fonseca B. A. (2004) One-step RT-PCR protocols improve the rate of dengue diagnosis com- pared to two-step RT-PCR approaches. J.
Clin Virol 30: 297 - 301.
9. De Paula S. O., de Melo Lima C, Torres M. P.. Pereira M. R., Lopes da l onseca B. A. (2004) One-step RT-PCR protocols improve the rate of dengue diagnosis com- pared to two-step RT-PCR approaches. J.
Clin Virol 30: 297 - 301.
DETECTION OF CHIKUNGUNYA VIRUS GENOME IN HEMORRHAGIC FEVER PATIENTS BY RT-PCR
Vu Xuan Nghia, Nguyen Linh Toan, Nguyen Trong Vien, Hoang Van Luong Military Medical University, Hanoi
Chikungunya virus (CHIKV) has re- cently caused hemorrhagic fever in Southeast Asian and African countries. Serological test may be used for diagnosis of CHIKV. but rapid detection of CIIIKV-RNA in clinical sample still need to be improved. In this study, we have described a direct RT PCR (one step) for amplification of El gene, in order to detect
CHIKV infection. There are 4 positive samples (8 %) among 50 tested clinical samples by RT- PCR w ith specific primer set with CHIKV. An- alyze and comparison sequencing the El gene of one positive sample, percentage nucleotide identity with CHIKV S27 to be 93%.
Key words: Hemorrhagic fever. Chi- kungunya, DENV
Tap chi Y hoc du phong. Tap XX. so 6(114) IS"
