Phát triển kỹ thuật Lamp (Loop-Media Ted Isothermal Amplification) cho việc phát hiện nhanh và chính xác vi khuẩn Escherichia Coli O157:H7

TAP CHI SINI-I HOC 2012, 34(3), 343-346

PHAT TRIEN KY THUAT LAMP (LOOP-MEDIATED ISOTHERMAL

AMPLIFICATION) CHO Vl£C PHAT HT$N NHANH VA CHINH XAC

VI KHUAN Escherichia coli 0157: H7

Hoang Phii Hi^p', Le Quang Huin^*

'"Dai hpc Su pham Thai Nguyen

'^'Vien Cdng nghe sinh hpc, huanlequang(ggmail.com

TOM TAT: Vi khuan Escherichia coh 0157:H7 la nguyen nhan quan trpng gay nen cac vu ngp doc thuc

pham va gay chet ngudi tren toan the gidi, M^t kj thuat phat hi?n nhanh vi chinh xac la rat quan trpng,

Shiga toxin 2 (ma hoa bdi gen stx2) la mpt lac nhan quan trpng gay ciia dong STEC noi chung va £ coli

0157:H7 ndi rieng, do v$y gen st2 ta mpt trong nhihig gen dich cho rat nhicu cac ky thual phat hien sir

d\uig, Trong nghien ciiu niy, chiing tdi da piiat trien kj thugi LAMP de phit hien nhanh va chinh xac

chiing vi khuan Escherichia coli 0157 H7. K^ ihu^t nay cd uu diem la phat hi?n nhanh, chinh xac va thiel

bj don gian. Bp mdi dupe tliict ke d|c bi^t dya tren trinh ty gen stx2 Chiing toi nhan ihay khong co su

khac nhau khi sii dyng duy nhat moi trong (BIP/FIP) hoiic ca 4 moi, Phan v'mg duong tinh dupe quan sat

bang mat thudng khi sii dung SYBR green ho^c dudi tia UV.

Tir khoa: Escherichia coh 0157: H7, LAMP, stx2 gen, FIP, BIP.

M6DAU

Escherichia coli 0157:H7 (£, coli

0157:H7) thudc nhdm vi khuan tao dpc td

Shiga (Shiga toxin-producing Escherichia coli,

STEC). Vi khuin E. coli 0157:H7 la mdt trong

nhiing nguyen nhan chinh nen cac vu ngd dpc

thuc pham va mpt sd benh d ngudi nhu: tieu

chay, viem rupt, hdi chiing sung huyet va suy

than (Hemolytic Uremic Syndrome, HUS) [2].

Theo Rasekh et al. (2005) [5], cd khoang 1,8

trieu ngudi chet vi hdi chiing sung huyet trong

khi sd ngudi td vong do ngd doc thuc pham

chua thdng ke dupe.

Viec phat hien nhanh E. coli gay benh la rat

quan trpng, vi vay, mdt phuong phip phat hien

chinh xac va hieu qui la rat cin thict. Hien nay,

viec phat hien nhan td gay benh thudng dua tren

CO che va dpc td gay benh cua cac chung vi

khuin. Ky thy^t LAMP li phuong phap nhan

ban DNA nhanh dupe phit trien bdi Notomi et

al. (2000) [4, 9], kj thuat nay_ dupe irng dung de

phat hien virus, vi khuan, nim vi ky sinh vat.

Phuong phap nay yeu cau mdt bd mdi d|c biet

dd nhan ra tir 2- 6 viing tren gen dich, do vay

lam tang dp nhay cung nhu dp nhanh ciia phan

ling. Ket qua cua phan ling LAMP c6 the dupe

quan sat bang mit thudng hay bang dien di yen

gel agarose hoac them thudc nhupm phat quang

dudi tia UV. Ky thuat LAMP dupe thuc hien

trong dieu kieu dang nhiet vi vay, chi can su

dung cic thiet bj giii nhiet nhu be dn nhiel, tii

im. Hon niia, thanh phan phin ling cd the bao

tdn trong mdt thing khi giii d nhiet dp 37''C tdt

hon de xuat ciia nha san xual [7]. Vdi nhung uu

diem do, ky thuat LAMP trd thanh ky Ihual

duoc sii dung rdng rai trong cic phuong phap

phat hien nhanh va chinh xac.

Da cd mdt vai nghien ciiu ky thuat LAMP

dd phit hien vi khuan E. coli 0157:H7 va mdt

sd vi khuan dpc mang gen slx2 dupe nghien cihi

tren thd gidi [1, 3, 8], Tuy nhien, d Vict Nam

chua cd nghien ciiu nao dupe cdng bo. Trong

nghien ciiu niy, chiing tdi sii dung ky thuat

LAMP cho viec phat hien nhanh vi chinh xac

chiing vi khuin E. coli 0157:H7 tai Viet Nam

nhim tao tien dd cho viec phit trien kit thii

nhanh vi khuin E. coli 0157:H7 trong thyc

phim ciing nhu cic mau nghien ciiu khac.

PHU'ONG PHAP NGHIEN CUU

Vat lieu

Vi khuin E. coli 0157:H7, chiing nay duoc

cung cip tir bp mdn Vi sinh vat, Dai hpc Khoa

hpc ty nhien, Dai hpc Qudc gia Ha Ndi.

Enzyme Bst polymerase,^ dem, dNTP ciia

hang Biolabs; cac cap mdi duoc dat ciia

invitrogen tdng hpp.