ENTEROBACTER SAKAZAKII

Enterobacter sakazakii

Enterobacter sakazakii MI HIM HA I VI TR S SINH

MC LC

DANH MC CC T VIT TT

BPW = Buffer Pepton Water EE broth = Enterobacteriaceae Enrichment broth FDA = Food and Drug Administration IFM = Infant Fomula Milks IAC = Internal Amplification Control MRS = DeMann Rogosa Sharpe Agar ompA = outer membrane protein A PCA = Plate Count Agar PCR = Polymerase Chain Reaction TSA = Trypticase Soya Agar

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Enterobacter sakazakiiVRBGA = Violet Red Bile Glucose Agar

CHNG I: TNG QUAN V TC NHN GY NG C1.1 Lch s pht hin: Theo c tnh ca T chc Y t Th gii (WHO) mi nm c hng triu trng hp bnh do thc phm xy ra trn ton th gii. Nm 1958 Enterobacter sakazakii ln u tin lin quan n mt trng hp vim mng no tr s sinh. K t , khong 70 trng hp nhim E. sakazakii c bo co, nhng ch c th l s trng hp ng k di bo co trong tt c cc nc v t l ny c th l cao hn. Lc by gi, sinh vt ny c gi l sc t mu vng - Enterobacter cloacae mi n nm 1980 mi c i tn l Enterobacter sakazakii (bi Farmer v cng s). Mt cuc iu tra quy m v cc sn phm sa bt v thc n kh cho tr din ra trn phm vi 7 quc gia chu u, M, Hn Quc v Nam Phi. Hn 200 mu t 110 sn phm khc nhau c phn tch cn thn truy lng du vt ca cc loi khun gy hi. Kt qu hon ton bt ng, 8 trong s 82 mu sa bt dnh cho tr s sinh c cha cc loi vi khun gy bnh ng rut. 12 trong 49 thc phm kh cng tnh trng tng t. Trong s cc

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Enterobacter sakazakiiloi khun c pht hin, ngi ta nhn dng c 13 dng vi khun thuc h Enterobacteriaceae gy bnh vim d dy - rut, trong c c Enterobacter sakazakii gy vim mng no. Vo nm 2001, mt thm cnh din ra ti khu chm sc c bit cho tr s sinh bang Tennessee (M), - hng chc b b vim mng no. Sau ny, ngi ta mi pht hin ra nguyn nhn l t mt l sa bt dnh cho tr. Nm 2004, cc chuyn gia ca T chc Lng thc v Nng nghip ca Lin hp quc (FAO) v T chc Y t Th gii (WHO) hp v pht trin hng dn quc t qua cc thng ip gio dc v Enterobacter sakazakii. 1.2 V tr phn loi: -Kingdom (gii): -Phylum (ngnh): -Class (lp): -Order (b): -Family (h): -Genus (chi): -Species (loi): 1.3 c im: 1.3.1. c im chung: Enterobacter sakazakii l trc khun hnh que, gram m, c kch thc chiu di l 3 micromet v chiu rng l 1 micromet, k kh tu nghi, khng c s hnh thnh bo t, c sc t vng, chu c p sut thm thu cao, di ng, cung cp cc vin nang v c vnh lng rung. Ngoi ra n thuc loi vi khun a nhit n ha. - Nhit ti a ca s pht trin: 41-45oC - Nhit ti thiu ca s pht trin: 5.5-8.0oC - Khng pht trin nhit : 4oC Bacteria Proteobacteria Gamma Proteobacteria Enterobacteriales Enterobacteriaceae Enterobacter Enterobacter sakazakii

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Enterobacter sakazakii1.3.2. c im sinh ha: Cc chng Enterobacter u c s hot ng ca -glucosidase v phosphoamidase. Enterobacter sakazakii sau khi phn lp thy c s ln men ca ng sucrose, raffinose, v -methyl-D-glucozit, nhng khng phi D-sorbitol, dulcitol, adonitol, Daeabinol. 1.4 Cu trc: - Enterobacter sakazakii c cu trc gen ompA vi trnh t ATCC 51329. 1.5. Yu t c lc: E.sakazakii tit ra c t enterotoxin (c t v ng rut). y l c t protein c trng lng phn t thp, thng c nhit n nh v tan trong nc. c t thng xuyn gy c t bo v git cht t bo bng cch thay i tnh thm ca mng t bo nh (biu m) t bo nim mc ca thnh rut.

1.6. C ch gy bnh: Trong t bo m ca ng vt c v, Enterobacter sakazakii c th gn vo ng rut ca t bo v tn ti bn trong cc i thc bo. Tuy nhin, c ch m adhesions ca Enterobacter sakazakii c kh nng th th trn t bo ch th hin nay qu trnh ny vn cha c xc nh r rng. Chng Enterobacter sakazakii c th sinh ra cc vin nang, nh c th tn cng vo c th t bo vt ch m vn trnh c hin tng i thc bo trong rut. Hn th na, vin nang ny gip bo v t bo trc khun Enterobacter sakazakii, to iu kin thun li gip n tn ti trong mi trng kh. Sau khi xm nhp vo t bo ch (nht l i vi tr s sinh) sinh vt ny s gn vo b mt mng plastics v lp silicon, v c nh th pht trin bi lp mng biofim. Vic nhim E. sakazakii i vi tr s sinh d xy ra nht trong qu trnh pha ch sa cho tr, trc khun ny s tn ti vi mt s lng ln di nm v ca bnh sa. Trong t bo ch, n c bao bc bi mng biofim c th trnh c nhng thay i bn trong t bo ch lm nh hng n sinh vt ny, t kh nng khng sinh ca n cng c pht trin. 1.7. Bnh v cc triu chng bnh: 1.7.1. Bnh:

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Enterobacter sakazakiiEnterobacter sakazakii l trc khun hnh que Gram m him khi gy bnh cho ngi ln. N ch yu gy bnh cho tr nh. Cc nghin cu ch ra rng tr s sinh, hoc nhng tr v thnh nin khc c nhng mc khng bnh khc nhau. Hu ht cc bo co u cho rng cc trng hp nhim khun u gy bnh vim rut, nhim trng v nhim khun ng huyt, vim mng no.Vic tn hi h thn kinh v t l t vong theo bo co ln n 40-80%. 1.7.2. Cc triu chng bnh: Triu chng thng thng tr: n ung km Kh chu Vng da Kh th Nhit khng n nh

Du hiu ca bnh vim mng no tr s sinh bao gm: St cao Qu bun ng hoc kh chu Tnh trng tr tr Bing n Mt li ra ch mm trn nh u cng ca c th v c ng kinh

1.8. Cc bin php phng v x l bnh: - i vi tr s sinh di 1 tui th bin php tt nht l cho b b bng sa m. Vic ny c xem l iu l tng nht chm sc tr s sinh. - Trong trng hp ngi m v mt l do no khng trc tip cho con dng sa th bn thn h phi c trang b y nhng kin thc v cch cho tr s dng sa ngoi c th chun b v s dng ng cng thc ca sa khi ri khi bnh vin, khi cn thit. - Nh nhng bo co gn y bi c quan chnh ph M FDA cho rng Enterobacter sakazakii khng chu nhit v c th d dng git cht nhit trn 60 0 , nh p dng

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Enterobacter sakazakiitrong cc qu trnh cng nghip, v d nh phng php tit trng Pasteur. V vy trc khi cho tr dng sa cn tin hnh kh trng dng c pha ch, un si nc dng (nhit trn 70oC) pha ch sa hoc un hon nguyn m bo cc iu kin an ton. - Nhng chuyn gia v y t cn c o to vi cht lng cao v c hng dn qun l ri ro hiu qu, m bo nhng iu kin v sinh ti u cho vic lu tr, x l, sn xut v chun b cc cng thc sa bt vi mc ch ngn chn s hin din v pht trin ca mt s loi vi khun gy bnh c trong cng thc sa c bit l Enterobacter sakazakii. -Cc trung tm chun on, phng tr v cha tr bnh nh bnh vin, cng phi m bo v sinh tt trong khu vc m bo ca h, bao gm c vic iu tr chng vi khun trc khi chun b cc cng thc v phi hp n lc gim thiu thi gian gia chun b v tiu dng. - V pha cc nh sn xut thc phm cho tr s sinh cn c bin php m rng cp d nh my m bo mc cao nht v cht lng sn phm v an ton. thc hin c yu cu ny cc nh sn xut phi: +C mt quy trnh kim sot nghim ngt nhm ngn nga nhim t khu sn xut n cc im mua hng ca ngi tiu dng. + Gim st cht ch cc thnh phn, c bit l nhng gia tng sau bc cui cng ca giai on ch bin nhit. +C cc bin php pht hin, loi b hay x l tt nht gim thiu s c mt ca Enterobacteriaceae hoc coliform trong mi trng sn xut. +o to, bi dng v t chc cn b m bo tun th nhng tiu chun an ton trong quy trnh cng ngh sn xut. +Kim tra tiu chun vi sinh ca thnh phm nhm m bo cht lng v s an ton ca sn phm. Nu t yu cu, tiu chun s c phn phi a ra th trng, cn khng phi x l hoc hy b trnh nh hng n sc khe ca ngi tiu dng.

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CHNG II : CC PHNG PHP XC NH2.1. Phng php truyn thng: Nhm chng ti s dng 2 phng php: nui cy v m khun lc.

Phng php nui cy:2.1.1 Nguyn tc: Ch s dng cc thuc th thuc loi phn tch, nc c s dng phi l nc ct, nc loi khong hoc c cht lng tng ng. Nc c s dng khng cha cc cht gy c ch s pht trin ca vi sinh vt trong iu kin th nghim. tng ti lp ca cc kt qu, nn s dng cc thnh phn c bn kh hoc mi trng hon chnh kh chun b mi trng nui cy. Cc gi tr pH c quy v 25 0 C. Dng acid clohydric [c(HCL)= 1mol/l] hoc dung dch natri hydroxit [c(NaOH)= 1mol/l] chnh pH nu cn. Mi trng nui cy v thuc th chun b nu chua s dng ngay th phi c bo qun trong cc iu kin khng lm thay i thnh phn ca chng, ni ti, nhit t 00

C n 5 0 C khng qu 1 thng , tr khi c quy nh khc. 2.1.2 Mi trng nui cy v thuc th: 2.1.2.1 Mi trng nui cy:

-Mi trng pha long mu nc m pepton Buffer Pepton Water (BPW)

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Enterobacter sakazakii-Mi trng thch u tng trypton (TSA) -Canh thang tryptoza lauryl sunfat ci bin (mLST)/mi trng vancomyxin -Mi trng thch phn lp Enterobacter sakazakii(ESIA TM ) -Mi trng vi sinh Brilliance Enterobacter sakazakii Agar (DFI) 2.1.2.2 Mi trng v thuc th v c tnh sinh ha: -Thuc th pht hin oxidaza -Mi trng L-lysin tch nhm carboxyl (decarboxylation) -Mi trng L-Ornithin tch nhm carboxyl (decarboxylation) -Mi trng L-Arginin cng hai hydro (dihydrolation) -Mi trng ln men hydrat carbon (nc pepton c m phenol, D-sorbitol, L-sobitol, L-rhamnoza, D-sucroza, D-melibioza v amygdalin) -Mi trng Simmons xitrat 2.1.3 Thit b, dng c: -Thit b kh trng kh (t sy) hoc thit b kh trng t (Autoclave) -Pipet 1ml -Ni cch thy, c th duy tr nhit 44 0 C 0.5 0 C -a Petri, ng knh t 90mm n 100mm -ng nghim, ng knh 18mm v di 160mm -T m, c th duy tr nhit 25 0 C 1 0 C, 30 0 C 1 0 C v 44 0 C 1 0 C tng ng -Vng cy, ng knh 3mm hay vng cy s dng mt ln -My o pH, c th o chnh xc n 0.1 pH nhit 25 0 C 1 0 C 2.1.4 Quy trnh phn tch:

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Chun b mu th/tin tng sinh: Cn 25g mu cho vo 225ml mi trng BPW

37C trong 18h 2h

Tng sinh chn lc trong mi trng vancomyxin/mLST: Chuyn 0.1ml t BPW cy vo 10ml mi trng vancomyxin/mLST 44C trong 24h 2h

Phn lp trn thch sinh mu chn lc: Cy vch mt vng y dch cy t mi trng vancomyxin/mLST ln thch sinh mu trong a Petri

44C trong 24h 2h

Khng nh: Sinh mu vng Chn nm khun lc in hnh v cy vch ln a TSA 25C trong 48h 4h

Khng nh: Sinh ha Chn mt khun lac mu vng t mi a TSA th sinh ha

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2.1.5 Thuyt minh quy trnh: 2.1.5.1 Chun b mu: Cn 9g mu th cho vo 225ml mi trng BPW. yn cho cc mu kh tan trong dch lng m khng khuy trn. nu sau 30 pht m mu khng ht th trn nh nhng mu vi mi trng. 2.1.5.2 Tin tng sinh: mu cy trong mi trng tin tng sinh 37 0 C 1 0 C trong 18h 2h 2.1.5.3 Tng sinh chn lc: Sau khi mu th c cy trong mi trng tin tng sinh, chuyn 0,1ml dch cy thu c 10ml mi trng vancomyxin/mLST. 44 0 C 0,5 0 C trong 24h 2h Nn s dng ni cch thy hoc t m kh cng bc m bo rng nhit ti a khng vt qu. 2.1.5.4 Phn lp Enterobacter sakazakiigi nh: Sau khi trong mi trng vancomyxin/mLST cy, ria cy mt vng y (khong 10l) ln b mt a thch phn lp. a 44 0 C 1 0 C trong 24h 2h. Sau khi , kim tra a thch sinh mu v s c mt hay khng c mt cc khun lc in hnh Enterobacter sakazakii gi nh. Cc khun lc in hnh c mu xanh l cy n mu xanh lc, c nh n trung bnh (1mm-3mm). Cc khun lc khng in hnh thng hoi trong sut v c mu tm. 2.1.5.5 Khng nh:

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Enterobacter sakazakiia. Sinh sc t vng Chn t mt n nm khun lc Enterobacter sakazakii gi nh in hnh kim tra trn a thch sinh mu . Cy vch cc khun chn ln b mt a thch TSA, sao cho sau khi quan st c cc khun lc tch bit. a ny 25 0 C 1 0 C trong 48h 4h. Sau khi , kim tra cc a TSA v s c mt cc khun lc mu vng. (Hnh 1)

Hnh 1. Khun lc mu vng pht trin trn mi trng TSA b. Khng nh bng sinh ha: C th s dng cc b kit th nh c bn sn trn th trng nhn dng bng sinh ha nhn dng Enterobacter sakazakii. Chn mt khun lc mu vng t mi a thch u tng trypton nhn dng bng sinh ha theo cc php th sau y: Php th oxidaza:

Dng kim cy s dng mt ln hoc que cy bng thy tinh, ly mt phn ca mi khun lc c trng chn.

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Enterobacter sakazakiiRia cy ln giy lc c lm m bng thuc th oxidaza. Khng s dng vng cy hoc que cy bng niken hoc crom. Nu mu ca giy lc khng i sang mu tm hoa c, mu tm hoc mu xanh m trong 10s th phn ng c xem l m tnh.(Hnh 2) Hnh 2. Kt qu php th oxidaza

Php th decarboxylaza L-Lysin: Dng vng cy, que cy hoc a thy tinh ly tng khun lc chn cy vo ngay di b mt mi trng lng L-Lysin decarboxylation. cc ng ny 30 0 C 1 0 C t 24h 2h. Sau khi c mu tm chng t phn ng dng tnh. Mu vng chng t m tnh. Php th decarboxylaza L-Ornithin: Dng vng cy, que cy hoc a thy tinh ly tng khun lc chn cy vo ngay di b mt mi trng lng L-Ornithin decarboxylation. cc ng ny 30 0 C 1 0 C t 24h 2h. Sau khi c mu tm chng t phn ng dng tnh. Mu vng chng t m tnh. Php th dihydrolaza L-Arginin: Dng vng cy, que cy hoc a thy tinh ly tng khun lc chn cy vo ngay di b mt mi trng lng dihydrolaza L-Arginin. cc ng ny 30 0 C 1 0 C t 24h 2h. Sau khi c mu tm chng t tnh. Mu vng (Hnh 3) phn ng dng chng t m tnh.

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Hnh 3. Kt qu ca php th decarboxylaza L-Lysin, L-Ornithing, L-Ariginin Php th ln men cc loi ng khc nhau: Dng vng cy, que cy hoc a thy tinh ly tng khun lc chn cy vo ngay di b mt mi trng lng ln men hydrat carbon. cc ng ny 30 0 C 1 0 C t 24h 2h. Sau khi c mu vng chng t phn ng dng tnh. Mu chng t m tnh. Php th s dng xitrat: Dng vng cy, que cy hoc a thy tinh ly tng khun lc chn cy vo ngay di b mt mi trng Simmons xitrat. cc ng ny 30 0 C 1 0 C t 24h 2h. Sau khi mi trng chuyn sang mu xanh chng t phn ng dng tnh.(Hnh 4)

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i chng trng

Phn ng m tnh

Phn ng dng tnh14

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Hnh 4. Kt qu ca th nghim xitrat 2.1.5.6 Din gii kt qu:

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Php th khng nh Sinh mu vng Oxidaza Decarboxylaza L-lysin Decarboxylaza L-Ornithin Dihydrolaza L-Arginin Axit sinh ra t vic -ln men D-sorbitol -ln men L-rhamnoza -ln men D-sucroza -ln men D-melibioza -ln men amygdalin -thy phn xitrat

Phn ng dng tnh hoc m tnh + + +

Phn trm chng Enterobacter sakazakii cho phn ng >99 >99 >99 90 >99

+ + + + +

95 >99 >99 >99 >99 >95

Phng php m khun lc:2.1.1. Nguyn tc:

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Enterobacter sakazakiiBrilliance Enterobacter sakazakii Agar (cng thc chun Chromogenic Enterobacter sakazakii Agar) l mi trng s dng cho vic phn lp v m Enterobacter sakazakii t mu thc phm.Cng thc * Tryptone Soya peptone Sodium chloride Ferric ammonium citrate Sodium desoxycholate Sodium thiosulphate Chromogen Agar pH 7.3 0.2 @ 25C gm/lt 15.0 5.0 5.0 1.0 1.0 1.0 0.1 15.0

2.1.2. Cch tin hnh: Cn 43.1g Brilliance Enterobacter sakazakii Agar vo mt lt nc ct. Lc u v un si n khi ha tan hon ton. Thanh trng bng ni hp 121C trong vng 15 pht lm ngui n 50C. Trn u v vo a Petri. 2.1.3. M t th nghim: Brilliance Enterobacter sakazakii Agar (DFI formulation1) da trn phn ng glucosidase , phn ng ny da trn s kt hp cht ch gia c cht chromogenic 5bromo-4-chloro-3-indolyl- -D-glucopyranoside c mt trong mi trng. Enzyme glucosidase, hin din trong Enterobacter sakazakii, c cht hydrolyses sinh ra khun lc mu xanh trn a mi trng mu vng xm. Proteus vulgaris cng c hot tnh -glucosidase dng tnh yu v cng c th pht trin sinh ra nhng khun lc c mu tng t nh E. sakazakii. Tuy nhin, trn mi trng ny, Proteus spp mc ln l nhng khun lc mu xm: chng sn sinh ra hydrogen sulphide trong s hin din ca ferric ions hnh thnh nn ferrous sulphide. Desoxycholate c tc dng lm n i s pht trin ca hu ht vi sinh vt Gram dng.

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Enterobacter sakazakiiPhng php FDA hin nay cho php pht hin Enterobacter sakazakii da trn vic sn sinh ra m mu vng theo nghin cu ca Muytjens et al.4. Cc mu c gi m trong nc qua m, sau c lm giu trong mi trng EE broth (CM0317), bc tip theo s dng mi trng VBRGA (CM0485) ng ha Enterobacteriaceae. 5 khun lc c chn lc v xp dy trn mi trng Tryptone Soya Agar (CM0131), gi m 3 ngy v quan st mu vng ca khun lc, n c trng cho Enterobacter sakazakii. (Hnh 5).Tuy nhin , phng php ny khng cho php phn lp Enterobacter sakazakii v vic kt hp EE broth vi VRBGA c th cho php cc dng khc ngoi Enterobacteria pht trin vt tri hn Enterobacter sakazakii ng thi cho kt qu m tnh sai. iu ny chng t rng khng th chn lc khun lc Enterobacter sakazakii t thch VRBGA. Hnh 5. Khun lc ca E.sakazakii 2.2 Phng php hin i(PCR): 2.2.1 Chun b mu th: - C tng cng 22 mu thc phm bao gm cc mu sa v cc sn phm thc n khc nhau cho tr s sinh ly t cc siu th a phng min nam Chu Phi. - i vi mu thc phm: t 100g sn phm vo bnh cha v trng v thm 900ml nc ct v trng nhit 45oC. Hn hp ny c qua m 37oC, sau khi c bn sao th ba th ly 2ml mu tch chit DNA. - i vi sn phm sa tit trng, cc bnh cha c trc tip qua m 37oC. Sau 10ml mu ny c chuyn vo canh trng EE v qua m 37 oC v khi mu c nhn ln ba ln th c ly chit xut DNA. 2.2.2 Tch chit DNA:

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Enterobacter sakazakiiTch DNA c thc hin theo phng php sa i ca Van Elsas. Trc khi chit xut DNA, 2ml mu c ly tm mc 5900 x g trong 10 pht. Sau khi ly tm ta loi b phn lng, ly phn cn ri b thm cc thnh phn sau: + ht thy tinh tit trng (0.6g) (c ng knh 0.2-0.3 mm) + 800 l m phosphat (1 phn 120 mM NaH2PO4 v 9 phn 120 mM Na2HPO4) + 700 l phenol + 100 l sodium dodecyl sulfate (SDS) 20%(m/v) Sau hn hp ny c ly tm trong 2 pht v nhit 60oC trong 20 pht v lp li bc tin hnh ny 2 ln. Sau khi , mu c ly tm trong 5 pht mc 1500 x g. Dch c thu li v cc protein c tch chit vi 600 l phenol. Tip theo ta cho 600 l hn hp phenol: chloroform: isoamylalcohol (25:24:1) cho n khi k trung gian c sch s. DNA c kt ta bng 0,1g sodium acetate 3M (NaOAc) (pH 5.5) v 0,6 g isopropanol. Cc mu c lu gi trong t lnh trong khong 1 gi v sau ly tm trong 10 pht 15000 x g. 2.2.3 Khuch i PCR: 2.2.3.1 Thit k mi: Cc cp mi Esak2 (5 'CCC GCA TCT CTG CAG TCT GAT C 3') v Esak3 (5 'CTA ATA CCG CAT AAC GTC TAC G 3') c pht trin cho khuch i c trng ca E. sakazakii. 2.2.3.2 Phn ng PCR: a) Chun b mu phn ng: - Phn ng pht hin PCR c thc hin trong tng th tch phn ng 25 l c cha 1U (0,35 l ) Taq ADN polymerase, 2,5 l m magie t do c cung cp vi enzyme, 2 mM (1,5 ml)MgCl2, 1 l dimethyl sulfoxide (DMSO), 1 mM (1 l ) dNTPs, 0.5 M (0,5 ml) mi mi v 750 g / ml (1 l ) mu DNA. - i vi mu i chng dng th ta ly 1 l chit xut DNA t mi trng tinh sch ca E. sakazakii v mu i chng m th ta thay th DNA mu bng 1 l nc ct v trng. b) Cch tin hnh PCR:

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Enterobacter sakazakiiPhn ng khuch i PCR c thc hin bng cch s dng mt Mastercycler (Eppendorf, c) v iu kin khuch i nh sau: - Hn hp phn ng c gi 95 C trong 2 pht cho cc bin tnh ban u, tip theo l 35 chu k trong 35 giy 95 C cho bin tnh thm. - 1 pht 61 C cho mi . - 1 pht ti 72 C ko di chui v chui cui cng ko di trong 10 pht 72 C. c) Kt cu ca IAC (internal amplification control): Mt phn ng PCR s dng cp mi Esak2 v Esak3 c thc hin trn DNA ly t t bo tinh sch E. sakazakii pht trin trn mi trng TSA. 10 l ca 850 bp PCR amplicon c phn gii vi 1 U (1 l ) ca cc enzym AluI endonuclease ct hn ch, vi s hin din ca 1 b m hn ch (1,5 l ) v 2,5 l nc ct v trng. Sau hn hp phn ng c qua m 37 C. Cc sn phm tiu ha c in di trn 3% (m / v) agarose gel c cha Ethidium bromide v biu din di tia UV. Hai kt qu phn on cha v tr lin kt ca cc mi tng ng c ct ra t gel agarose v lm sch bng cch s dng b sn phm lm trong PCR tinh sch cao theo c im k thut ca nh sn xut (Hnh 1).

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Sau khi lm sch, 10 l mi on c ni li khi s dng 1 U (1 ml) T4 DNA ligase. Mt phn ng khuch i PCR c thc hin trn on ni bng cch s dng PCR mi Esak2 v Esak3 nh m t. Cc kt qu Sn phm PCR c nhn bn v tnh bng cch s dng Inst / AcloneTM b sn phm PCR nhn bn. Cc t bo bin i c sng lc cc chn kch thc ng bng mi T7 (5'-GTA ATA ATA CGA CTC ACT GGG-3 ') v SP6 (5'-TAC GAT TTA GGT GAC ACT ATA G-3 '). PCR c thc hin trong mt tng th tch phn ng 50 l c cha 0,5 M (2l) ca mi mi, 0.5 mM (2l) dNTPs,1 U (1,5 ml) Taq ADN polymerase, b m cha MgCl2 (5 ml) cung cp vi cc enzyme v cc t bo chuyn i. Plasmid DNA c phn lp t cc chng ti t hp bng cch s dng Qiagen Midi plasmid kit. Cc DNA plasmid c s dng nh mt IAC trong tt c cc PCR phn ng pht hin E. sakazakii Nam Phi trong sa v thc n cho tr. Vic xy dng IAC c lp trnh t vi Esak3 mi bng cch s dng chui DNA ABI PRISM 377 ti cc c s trnh t DNA ti i hc Stellenbosch. iu ny c thc hin xc nhn s hin din ca v tr kt ni hai mi v kch thc chnh xc ca IAC.

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Enterobacter sakazakiiSau khi xc nh nng DNA ca DNA plasmid b c lp, nng ti u l to ra mt tn hiu dng tnh cho c hai E. sakazakii DNA v cc IAC c xc nh. Phn phn c ca 1 ml ca IAC c thm vo tt c cc phn ng PCR c thc hin, khng bao gm mu i chng dng v mu i chng m. 2.2.4 Kt qu PCR:

Sau khi ct cc cp base c kch thc 850 (bp) t sn phm PCR E. sakazakii, hai on cha cc v tr lin kt ca cc cp mi (79 v 161 bp kch thc) c gn vi mt IAC kch thc 240 bp. IAC c pht trin ngn nga kt qu m tnh gi (Hoorfar et al., 2004) v mt on dng tnh cho c hai DNA mc tiu (850 bp) v IAC (240 bp) vi nng ti thiu l 0,72 pg.ml-1 ca IAC c thm vo hn hp phn ng PCR. IAC c gi tr ngn chn kt qu m tnh gi v l mt ch th khi phn ng PCR tht bi. Trong kt qu ca th nghim ny, s hin din ca 850 bp sn phm khuch i PCR cho thy s c mt ca E. sakazakii, trong khi mt on 240 bp cho thy s khuch i PCR thnh cng.

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Enterobacter sakazakii Kt lun: Enterobacter sakazakii c pht hin trong 4 mu trn tng s 22 mu ( chim 18% sn phm th nghim)(Bng 1). iu ny chng minh s dng phng php PCR c hiu qu sau khi mu tng sinh trong canh trng EE. Nh vy trong tng lai phng php PCR kt hp vi IAC c th c dng nh l mt phng php nhanh chng v chnh xc pht hin E. sakazakii trong cc sn phm t IFM hn l phng php xc nh truyn thng nh hin nay.

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CHNG III. KT LUN V NGH3.1 Kt lun: S hin din ca Enterobacter sakazakii trong IFM nn c coi l mt nguy c nghim trng v tim nng ca vi sinh vt ny c nhn ln trong qu trnh chun b v bo qun sa trc khi tiu th cc sn phm sa hon nguyn (Anon., 2004). Enterobacter sakazakii l mt vi sinh vt mi trng v do phi khuyn co rng tt c cc nguyn liu th v cc sn phm cui cng phi c kim tra v nh gi cao v s hin din ca trc khun ny ngn chn s ly nhim c trong cc sn phm sa bt c phn phi. Thm ch vic nhim E. sakazakii vi t l thp vn c th gy ri ro cho tr s sinh, do , iu quan trng l phi gio dc mi ngi nht l nhng bc cha m v cch chm sc i vi tr s sinh v cc nguy c tim n lin quan n vic s dng khng ph hp ca IFM. 3.2 ngh: i vi bi bo co ny nhm chng ti gp mt t kh khn v vic tm kim ngun thng tin xc nh cu trc v c ch gy bnh ca loi ny cha c xc nh c th. Chng ti hy vng s c cung cp thm ti liu hon thnh tt hn bi bo co ny. Hin nay trn th gii vic nhim trng IFM bi Enterobacter sakazakii c bo co nhng Vit Nam th con s ny cha c thng k r rng. Chnh v th, nc ta nn c mt con s c th v s lng tr s sinh b nhim bi E. sakazakii nhm mc ch khuyn co n ngi tiu dng trong vic la chn sn phm IFM trong v ngoi nc. Vic sn xut sa ch quan tm n hm lng ch tiu cc cht dinh dng nh DHA, st, cc loi vitamin, protein, lipit m khng ch trng n ch tiu vi sinh (s hin din ca cc loi vi khun c hi, c bit l Enterobacter sakazakii) phn no lm nh hng n cht lng sa e da sc khe i vi tr s sinh.

CHNG IV. TI LIU THAM KHO

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Enterobacter sakazakii1- L Vn Mn(ch bin), Li Mai Hng - Th nghim vi sinh vt hc thc

phm- NXB H Quc Gia TPHCM2- Sa v sn phm sa- Pht hin Enterobacter sakazakii TCVN 7850:2008

3- Cc trang web tham kho:http://scholar.sun.ac.za/bitstream/handle/10019.1/3097/Kemp,%20F.pdf? sequence=1 http://www.iss.it/binary/publ/cont/275%20%20ANN_08_39%20Fiore.1224498732.pdf http://www.pjbs.org/pjnonline/fin1712.pdf http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563671/ http://journals.uzpi.cz/publicFiles/13828.pdf

http://nguyenanhvn.com/san-pham/moi-truong-vi-sinh-brilliance-enterobactersakazakii-agar-cong-thuc-dfi--1847.html

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ENTEROBACTER SAKAZAKII