Nguyen Thi Trung Tap chi KHOA HQC & CONG NGHE 164(04); 2 1 5 - 2 2 0
NGfflEN CUtJ KHA NANG NHAN BIET DAC HIEU CAC KHANG NGUYEN CUA Listeria monocytogenes CUA MQT SO KHANG THE DON DONG NHAM SU'DUNG TRONG TAO QUE THU'NHANH
Nguyen Thj Trung'
Vi^n Cdng nghe sink hoc, Viin Hdn lam Khoa hgc vd Cong nghe Viet Nam TOM TATListeria monocytogenes la vi khuan gay benh ngg dgc thuc pham, Vi khuin nay co the phat hi?n dugc bang nuoi cay trong moi trudng chgn Igc, phuong phap sinh hgc phan tu hoac phuong phap mien dich hgc. Cong trinh nay cong b6 ket qua sang lgc khang the de gan len mang trong phuang phap sac ky miSn dich nham phat hien L monocytogenes. Khang the don dong C86030M nhan biet duoc cac khang nguyen cua L monocytogenes gom protein tSng so, protein tan va protein ming ngoai dugc su dung de lam khang th6 biy bat Khang th^ don dong C86020M chi nhan biet protein ming ngoai cda L. monocytogenes mk khong nhin biSt protein mang ngoai cua E coli hoac Salmonella dugc su dung lam khing the phat hien. Cap khing the nay da dugc sii dung tao que nhung, ket qui la que nhiing da hogt dgng dugc. Ket qua ban dau nay hua hen se md ra vi?c san xuat dugc cac que thu nhanh de phat hign L. monocytogenes uong thuc pham.
Tir khoa: Listeria monocytogenese, khdng thi don dong. protein mdng ngoai. te bdo nguyen ven.
ngo doc thuc phdm
MO DAU
Mac du L. monocytogenes khong phai li mdt benh ph6 bien trong cpng ddng nhung nd la mgt tic nhan gay benh quan trgng d nhiing benh nhin mang thai, tre so sinh, ngudi gii va nhung ngudi bi suy giim mien dich. Dd li mOt logi vi sinh vit giy benh ngp dgc thuc pham dien hinh. Listeria con li mgt tac nhin Co the phan lip dugc Listeria trong dat, nude vi rac hihi ca dang phin huy. L.
monocytogenes cd the phat hien dugc bang phuang phap nudi cay truyen thdng, phucmg phap mien dich hgc, cim bien sinh hpc hogc cac phuang phap sinh hpc phin td [4], [11].
Cic phuang phap nudi cay truyen th6ng li phuang phap ding tin cay, chinh xac vi van dugc su dyng mac du chung tieu ton thdi gian vi cong sue [4]. Cic phuang phap dua vio thdng tin di truyen nhu PCR, giii trinh tu gen hogc lai DNA cin khoing 48 gid de dpc ket qua [11]. Cac phuong phap mien djch hpc nhu ELISA, EFLA can khoang 2 gid d6 co k6t qua, tuy nhanh han phuang phap nuoi ciy truySn th5ng nhung dp tin ciy cua chung con phy thugc vao cac khang nguyen vi cac khangthS dugc su dyng [13].
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Khing nguyen cda L. monocytogenes khi dan gian, cic khing the die hieu cda chung da dugc nghien ciiu va chung khdng phin ung cheo vdi cic khing the cda cic loai khac. Do vay, chung ta cd the sd dyng khang the hoac khang nguyen de tao cic bd sinh pham chin doin L. moneocytognes. Hien nay, co kha nhigu khing the dem ddng khang L.
monocytogenes da dugc san xuat. Khang the dan ddng khing protein p60 cda L.
monocytogenes (dong P6007) da dugc sin xuit bdi cdng ty Millipore. Khing the niy nhan dien protein mang ngoai cua L.
monocytogenes [7], Cdng ty Meridian sin xuit mdt sd loai khang the don dong khing L.
monocytogenes nhu C86020M, C86030M, C86713M, C86503M. Cac khing thg nay die hieu vdi protein ming ngoai hogc li te bio cdn nguyen vgn. Hoac khang the dan dong C86032M, C86909M chi nhan biSt protein mang ngoai cua L. monocytogenes [6], C6ng ty USBio sin xuit khang tha dan dong L2650-02P khang protein P60 cda L.
monocytogenes [8].
Sinh phim phit hien nhanh dua tren ca sd
phan ung khang nguyen-khang the tren mang
215
Nguyen Thj Trung Tap chi KHOA HQC & CONG NGHE 164(04): 215-220 mdng cdn dugc gpi li sac ky mien dich cd the
li dang thd nhanh hoic la dang que nhung dan giin. Chung dugc phat trien dya trgn nguyen I;^ cua phan ung ket dinh die hieu cua khang nguyen - khang thg tren mdt mang mdng lim bang giiy, nylon hogc nitrocellulose [13], De san xuit mdt que thd nhanh chan doan L. monocytogenes chung ta cin phai co hai loai khang the don dong cung nhin biet L. monocytogenes nhung khdng nhan biSt nhau, Mgt khang the gpi la khang thi bay bit dugc cd dinh tai cua s6 nhin miu.
Mgt khang the ggi la khang the phit hien dugc cd dinh tren vach kiem tra. Viec lua chpn dugc hai khing the nay la mpt viec lam t6n khi nhiSu thdi gian. Trong nghien ciiu nay chung t6i trinh bay cic ket qua nghien ciiu ve viec tim kiem cgp khang the dan dong thuang mai phu hgp de tao que thu nhanh phit hi^n L. monocytogenes. Ket qui li cap khang thS C86020M, C86030M da duac lya chpn de tao que thd nhanh phit hien L. monocytogenes. Que thd tao ra da dugc thd nghiem ddi vdi protein mang ngoii cda L. monocytogenes cho kSt qui tdt. Ca hai vgch ddi chung va vach kiem tra da xuat hien bang miu chdng to chung dam bao dugc tinh khing the trong thi nghiem tao que thd thd nghiSra.
VAT L1$U, PHUONG PHAP NGHIEN CUU Vat li^u va mdi trudng nudi cay Khdng the: Khing the dgn ddng khang L.
monocytogenes (IgM) san xuat td chupt C86020M; khang thi dan ddng khing L.
monocytogenes (IgGl) sin xuit tu chugt - C86030M (Meridian Life Science, My); khang the khang chugt (IgM) cgng hpp alkaline phosphatase A9688-1ML (Sigma, MJ).
Chitng vi sinh vgt: E. coli ATTC11105, S.
enteritidis ATTC13076 chung thd nghiem luu tru tai Vien Cdng ngh$ Sinh hpc; L.
monocytogenes do nhdm nghien ciiu cua GS.TS. Nguyin Thiiy Chau, Vien Bao quin sau thu hoach phin lip tai Viet Nam cung cip.
Cdc vgt lieu khdc: Bd kit hlgn mau ddng cho alkaline phosphatase - 170-6432, ming nitrocellulose (Bio-Rad, My).
Moi truang nuoi cdy: LB (5,0 g cao nim men;
5,0g prptone, 10,0 g NaCl, nude cit dd 1000 ml); LB A: 1000 ml moi hirdng LB b6 sung 15 g bdt thgch; BHIB - 299070 (BD): 7,7 g Calf Brains, 9,8 g Beef Heart, 10,0g peptone, 5,0g NaCl, 2,0 g Dextrose, 2,5 g Na2HP04, nude cit dd 1000 ml; BHIA - 241830 (BD): thanh phin tuang tu moi trudng BHIB, bd sung 15 g bdt thach cho mdi iOOO ml; tit ca cac mdi trudng dugc chuan bi, khu trung d 121°C, bao quan d 4''C va dung trong mdt tuan, Phuwng phap nghiSn cu-u Nuoi cay vi khuan
Nudi ciy vi khuin trong m6i trudng long:
Chuyin mgt khuan lac tu dTa mdi trudng thach vao mdi trudng long phu hgp, lie 200 vdng/phut td 12-16 gid dnhietdp thich hgp.
Tdch cdc logi protein
Phuang phip tich cac logi protein dugc thuc hien theo phuang phap cua Quan vi d6ng tic gia nam 2013 co cii tien [10].
Chudn bi mdu vi sinh; Cic vi khuin thi nghiem dugc nudi cay trong moi trudng long thich hgp qua dem d nhiet do thich hgp tiing loai. Ly tarn 6000 vong/phiit trong 7 phut de thu t6 bio, Te bio dugc nia 3 lin bing dimg dich 50 mM Tns pH7,5. Bio quan te bao d 4''C din khi sd dung.
Phi va tl bio bang sieu am: Hoa tl bio thu td 50 ml dich nudi cay vao 5 ml dung dich 50 mM Tris-HCl pH7,5, sieu am 3 phut vdi chu ki 10 giay boat dgng, 10 giay nghi.
Thu protein tong so: Dich te bio da si6u am dugc ly tam 2500 vdng/phdt trong 10 phut.
Phin dich noi chua protein tdng s6 cua tl bio dugc thu lgi.
Thu protein tan: Dich protein tdng sd dugc ly tam 13000 vdng/phut trong 60 phut. Phin dich noi chua cic protein tan cua tl bao dugc thu lai.
216
Nguyin Thj Trung Tap chi KHOA HQC & CONG NGHE 164(04): 2 1 5 - 2 2 0 Thu protein mdng ngoai- Phin can thu dugc
sau Idii phan tach protein tan dugc rda nhilu lin b i n g dung dich 50 n i M Tris-HCl pH 7,5.
Cin d u g c hda vao d u n g dich 0 , 1 % saccosyl trong 50 m M Tris-HCl pH7,5, l i e 60 phut 200 vong/phut d 4°C. L y t i m tdc dp 13000 vdng/phut trong 60 phut, loai bd p h i n dich noi chiia c i c protein mang trong, thu phan can chua protein m a n g ngoai.
Thi nghiem dot-ELISA
Thi nghiem dot-ELISA d u g c tien hanh theo mo ta cua Donald va ddng t i c g i i nam 2000 CO cii tien [2]:
Gan protein len mdng: 5 pi dung dich protein dugc nho true tiep len mdt vi tri tren mang nitrocellulose. 60 phdt sau, chuyen m i n g sang dung dich 5 % sda khong beo trong dung dich TBS (1000 ml: 25 ml 1 M Tris.HCl p H 7,5, 10 ml 5 M NaCl), l i e d 4''C qua dem. Rda ming b i n g dung djch T B S , 10 phut; sau do nia b i n g dung dich T T B S (IOOO ml T B S + 250 pi Tween 20), 5 phut; lap lai 3 lin.
Nhupm khdng the: N h u g m mang trong dung dich k h i n g the khang Listeria san xuat td te bao chugt, do pha loang thich hpp trong dung dich TBS d nhiet dp phong trong 3 gid. Rua mang bang T B S va T T B S n h u m 6 t i d tren.
Sau do, mang d u g c n h u p m tiep vdi dung dich khing the khang chudt cgng hgp alkaline phosphatase trong 3 gid d 37''C. Rda mang bing T B S va T T B S n h u tren.
Hten mdu: M i n g d u g c nhugm trong dung dich hign m i u den khi quan s i t thay cac c h i m mau hien len ro rang, d d n g p h i n u n g bang each rda mang nhieu lan vdi n u d e , quan sat va chyp anh.
Dipt di protein tren SDS-PAGE Di?n di d u g c t i l n h i n h theo p h u a n g phap cua Laemmli [9],
KET Q U A v A T H A O L U A N Tach cac loai p r o t e i n
Thuc hien tach c h i l t cac logi protein theo phuong p h a p cda Quan va ddng t i e gia [10]
n h u chdng tdi md ta chi tiet trong phan phuong phap. Ket q u i d Hinh 1 cho thay da thu d u g c c i c logi protein tdng sd, protein tan, protein mang ngoai cda vi khuan theo quy trinh md t i d p h i n p h u o n g phap. C i c protein mang t i c h d u g c rat nhieu doi vdi c i c vi k h u i n gram i m (dudng chgy 4-9), N h u n g protein mang thu dugc d vi khuan L.
monocytogenes kha it, d i e biet la protein m i n g n g o i i . Dieu nay hoan toan phd hgp vdi c i c cong bd trudc d i y ve su khac biet cua Idp vd te b i o gida vi khuan gram am v i vi k h u i n gram d u a n g . Cac vi khuan gram am cd Idp vd te b i o day, bao quanh la mgt lop mang ngoai, trong khi vt k h u i n gram duong co lop vo mdng, khdng cd Idp mang ngoai bao quanh [1]. Vi the protein mang ngoai t i c h chiet td L.
monocytogenes r i t it (dudng chgy 3).
I 2 3 M 4 5 6 M 7 8 9
kJ
Hinh L Phdn tich cdc phdn dognprotein tdch tir vi khudn bang dien di trin SDS-PAGE Dudng chay 1-3: protein t6ng so, protein tan va protein mang ngoai cua L monocytogenes, tucmg dng; 4-6- protein tong s6, protein tan va protein mang ngoai cua S enteritidis ATCC13076, tuong ung; 7-9' protein t6ng sl, protein tan vi protein mang ngoai cua E. coli ATCC11105, tuong ung, M: thang protein chuanThi nghiem dot ELISA
K h i n g t h i C86030M nhan b i l t tat ca cac loai khang nguyen cua L. monocytogenes dem k i i m tra gom protein tdng so, protein tan va protein mang ngoai (Hinh 2), Khang t h i nay khdng nhan b i l t protein mang ngoai cua cac vi khuan Gram am ciing gay benh ngp dpc 217
Nguyen Thi Trung Tap chi KHOA HOC & C 6 N G NGHE 164(04): 2 1 5 - 2 2 0 thuc p h i m nhu S. enteritidis ATCC13076, E.
coli A T C C l 1105. D i l u nay k h i phu h g p vdi khuyen c i o cua n h i s i n xuat rang k h i n g the C86030M nhgn biet p h i n doan protein m i n g ngoai cda L. monocytogenes hogc te bao L.
monocytogenes nguyen ven, Khang the n i y khdng phan iing cheo vdi E. coli. Salmonella [5]. Qua kSt q u i thi nghiem nay, khang the C86030M d u g c chgn lam k h i n g thS bay b i t khi gan len mang que nhung p h i t hien L.
monocytogenes. B i n g each n i y tat ca cac k h i n g nguyen cua L. monocytogenes d u g c b i t lgi khi di chuyen qua vi tri g i n khang the,
I 2 3
Hinh 2. Su nhgn biit ciia khdng thi C86030M doi vdi cdc protein bang dot ELISA Dudng chgy 1: protein cua E. coli ATCCl 115; 3:
protein cua S enteritidis ATCC13076; 3: protein cuaZ. monocytogenes.
1 2 3
Hinh 3. Sif nhdn biit cua khdng the C86020M ddi v&i cdc logi protein duac kiim tra bdng dot ELISA Duong chay 1: cic loai protein cua L.
monocytogenes; 2: cac loai protein cua E. coli ATCCl 115; 3 cac loai protein cua S. enteritidis ATCC13076.
218
K i t q u i d Hinh 3 cho t h i y khang t h i C86020M chi nhan bilt protein mang ngoii cda L.
monocytogenes. N h u n g khang the nay nhan biet protein tan v i protein t i n g sd cua t i t ca c i c m i u kiim tra gdm E. coli va Salmonella. Neu L monocytogenes dem kiSm tra tdn tai d trang thai nguyen ven hogc chi su dung protein mang ngoai cho cac thu nghiem m i i n dich thi s6 thu dugc k i t q u i d i e hiSu. K h i n g t h i C86020M dugc dung lam k h i n g the phit hifin, Khang the nay dugc ^ n vdi hat nano vang de lam chat chi thi mau tren que thu.
Thu nghiem que nhiing diing khdng the bay bat C86030M vd khdng thS phdt Men C86020M
Quy trinh tao que nhiing khdng d u g c trinh bay chi tiet trong c6ng trinh nay. Trong dd, khang the C86030M su dung l i m khang the bay bat d u g c gan tgi vach kiem tra; khang the C86020M cdng hgp hat nano v i n g su dyng l i m k h i n g the phat hien d u g c gan tgi vi tri nhan mau; khang the khang chugt dugc gin tai vgch doi chung.
vach kiem tra Vach d6i chiing
Viii^nhan mau Hinh 4. Que nhiing gdn cdp klidng thi C86050M vd C86020Mthtinghiim v&iprolein mdngngoai
cita L monocytogenes Ket q u i n h i n dugc cho thay co 2 vach mau xuat hien tgi cda sd d p c ket q u i . D i l u n i y chiing td khang t h i C86020M (IgM) cdng hgp hgt nano v i n g da ket hgp d u g c vdi khang nguyen m i n g n g o i i cua L. monocytogenes va p h d c hgp nay da di chuyen d i n vj tri g i n k h i n g t h i C86030M ( I g G l ) . Tai d i y khang t h i C86O30M ( I g G l ) lien k i t vdi protein mang n g o i i cua L. monocytogenes trong phuc C86020M (IgM)-khang nguyen v i bj giii lai tao thanh bang mau. Nhiing khang t h i C86020M (IgM) khdng g i n khang nguyen hogc phdc C86020M (IgM)-khang nguyen khong hi gid lai d vj tri kiem tra se t i l p tyc di
Nguyin Thi Tning Tap chi KHOA HQC & CONG NGHE 164(04): 215-220 chuyin din vi tri gin khang thi khang chugt
(IgM), Tgi day khang thi C86020M (IgM) se lien kit vdi khang thi khing chuot (IgM) va tao thanh bang mau. Budc diu cho thiy que nhung hoat dgng tdt ddi vdi khang nguyen mang ngoai cua L. monocytogenes. Kit qui ban dau nay can tiep tuc dugc kiem chdng, danh gii dg nhay, dd dac hieu, kha nang phit hien dugc L. monocytogenes d dgng te bao, trong hon hgp mlu va trong miu thyc phim, KET LUAN
Qua cac ket qui dat dugc chung tdi nit ra kit tuan ring cd the sd dung cap khang the C86030M, C86020M dl tgo que nhdng phat hien L. monocytogenes. Trong dd, khing thi C86030M dugc sd dyng lim khing thi bay bat va khing thi C86020M dugc su dyng lam khang thi phat hign. Cap khing thi nay da dugc sd dung dl tao que nhung v i phat hien dugc protein mang ngoii cua vi khuin L.
monocytogenes.
Lai cdm an: Nghien cuu dugc thuc hign tgi Phong thi nghigm trgng dilm C6ng nghg gen, Vign Han lim Khoa hpc vi Cdng nghe Viet Nam va sy trg giup \\i Hpc vien Quan Y.
TAI LIEU THAM K H A O 1. Brown L,, Julie M. W., Prados-Rosales R., Casadevall A, (2015), "Through the wall:
extracellular vesicles in Gram-positive bacteria, mycobacteria and fiingi", Nature Reviews Microbiology. 13, pp. 620-630.
2. Donald G. G., Christopher J. F., Goodhart S A (2000), "The dot blot ELISA' A rapid and simple experiment to demonstrate antibody- antigen interactions". The American Biology Teacher, 62 (8), pp. 583-587,
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"Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes; a review", FEMS Microbiology Reviews. 29, pp.
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(2010), "Human listeriosis caused by Listeria ivanovii". Emerging Infectious Diseases 16(1), pp.
136-138.
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9. LaemmHU K (1970), "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature 227, pp. 680-685.
10. Quan S,, Annie H., Jean-Frangois C , James C, A, B, (2013), "Isolation of bacteria envelope proteins, Anne H Delcour (ed.). Bacterial cell surfaces: Methods and Protocols", Methods in Molecular Biology, 966, pp. 359-366.
II.Rodriguez-Lazaro D., Hernandez M. (2006),
"Molecular methodology in food microbiology diagnostics; trends and current challenges", lUFoST World Congress 13' World Congress of Food Science and Technology, pp. 1085-1099.
I2.Salimnia H., Patel D., Lephari P. R,, Fairfax M R , Chandrasekar P. H (2<:>\0), "Listeria grayi:
vancomycin-resislant, gram-positive rod causing bacteremia in a stem cell transplant recipient", Transplant Infectious Disease. 12(6), pp. 526-528.
13.Singer J. M., Plolz C. M. (1956), "The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis", American Journal of Medicine. 21, pp. 888-892.
Nguyen Thi Trung Tap chi KHOA HOC & CONG NGHg 164(04): 2 1 5 -
S U M M A R Y
STUDY ON T H E ABILITY O F SPECIFICITY R E C O G N I Z I N G L I S T E R I A M O N O C Y T O G E N E S ' A N T I G E N S OF S O M E M O N O C L O N A L A N T I B O D I E S F O R USE IN P R O D U C I N G T H E Q U I K S T I C K TEST
Nguyen Thi Trung Institute of Biotechnology, Vietnamese Academy of Science and Technology Listeria monocytogenes is a bacteria that causes food poisoning. This microorganism can bt detected by culture in a selective medium, molecular biology or immunology methods. In thij study, the results of screening antibodies for use attached to the membrane of immune chromatography is published. Accordingly, the monoclonal antibody named C86030M which \i recognized antigens of L, monocytogenes including total, soluble and outer membrane proteins are severed as a capture antibody. And the monoclonal antibodies named C86020M, which is onlj recognized the outer protein membrane of L. monocytogenes without E. coli or Salmonella is usee as a detection antibody. Two monoclonal antibodies were used to create dipsticks and the dipstick;
is already well-done. This initial results promise to open up the production of the test strips tc detect L. monocytogenes in food.
K-tyworAi: Listeria monocytogenese, monoclonal antibody, outer membrane protein, intact cell food-poisoning
Ngdy nhgn bdi: 06/01/2017; Ngdyphdn biin: 09/02/2017; Ngdy duyit dang: 27/4/2017
Tel-0936 270978, Email trungnguyen 220
