Phn 1

M UNg c thc phm c th xy ra khi chng ta s dng thc n, nc ung nhim khun hoc ha cht c hi... Cc nghin cu gn y xc nh c mt trong nhng nguyn nhn ca nhiu trng hp ng c thc n l do vi khun Escherichia coliO157 (E. coli O157).Vi khunE. coli O157 thuc nhm vi trng ng rut Enterobacteriaceae, c trong ng rut ca ngi v gia sc chng c tm thy i trng nn cn gi l vi khun i trng. Trong t nhin, vi khun E.coli O157 c th c tm thy trong rut v trong phn ca cc loi gia sc, c bit l trong phn b, sa cha kh trng Cc bnh c lin quan n vi khunE.coli O157 l bnh tiu chy, vim ng nc tiu v mt s bnh khc. Triu chng b nhim trng vi khun thng khc nhau, nhng nhn chung, bnh nhn c cc triu chng nh: nn, au bng, st, tiu chy thng c muCc chuyn gia c tnh rng M mi nm c khong 70 ngn ngi mc bnh lin quan nE. coli O157. S v ng c thc phm do vi khun ny gy ra ng th 3 sau cc nguyn nhn do Campylobacter v Samonella.Ti Vit Nam, theo s liu Cc thng k t nm 2007 n nm 2013 cho thy trung bnh mi nm c khong 234 v ng c thc phm xy ra vi khong 8.211,3 ngi mc v khong 48,3 ngi cht. T l b ng c thc phm trung bnh l 7,05/100.000 dn, t l cht l 0,06/100.000 dn/nm. Hin nay, cc nh khoa hc tm ra c nhng bin php cha tr bnh do E. coli O157 gy ra, cng nh cc phng php pht hin sm s c mt ca n trong thc phm c bin php x l kp thi nhm ngn chn v hn ch cc s c v an ton thc phm c th xy ra.Phn 2

TNG QUAN V VI KHUN E. COLI O1572.1. Tng quan v E.coli O157

2.1.1. Hnh thi v c tnh sinh vt, ha hc ca vi khun E. coli O157Vi khun E.coli O157 c dng hnh que nh, hai u trn, ng ring r, i khi xp thnh chui ngn (hnh 2.1) v bt mu Gram m (mu hng); c kh nng di ng.

Hnh 2.1. Hnh dng E.coli O157

Bng 2.1. Mt s c tnh sinh hc, ho hc ca vi khun E. coli O157TTc tnh sinh haDng tnh (+)/ m tnh (-)

1Gram (-)(+)

2Lactose (+)

3Glucose(+)

4H2S(-)

5Kh nng sinh kh(+)

6Kh nng di ng(+)

7Mannitol(+)

8Urease(-)

9Indol(+)

10Sorbitol(-)

2.1.2. c t v c ch gy bnhVi khun E.coli O157 thng gy mt s hi chng nguy him ngi nh: vim kt trng xut huyt (hemorrhagic colitis), ur huyt (hemolytic uremic syndrome), suy thn; thm ch c th dn n t vong. Kh nng gy bnh ngi ca E.coli O157 cn l kt qu ca kh nng sn xut nhiu yu t c lc, ng ch nht l cc c t Shiga (Stx), l mt trong nhng cht c mnh nht c bit i vi ngi. Shiga toxin c nhiu bin th (Stx1, Stx2, Stx2c) v hot ng nh cc cht c bng cch c ch s tng hp protein trong t bo ni mc v cc t bo khc. Ngoi Shiga toxin, E.coli O157 sn sinh nhiu yu t c lc gi nh khc, bao gm c protein m h tr trong vic gn kt v tng kch thc khun lc trong rut v c th gn kt cc t bo mu v gii phng st gip h tr cho qu trnh trao i cht ca E.coli O157.Phn 3PHNG PHP PHT HIN VI KHUN E.COLI O157 THEO TIU CHUN ISO 16654:20013.1. Nguyn tcCc vi sinh vt hnh thnh cc khun lc in hnh trn mi trng thch a c s dng trong tiu chun ny, c sinh indol v dnh kt c hiu vi khng huyt thanh khng khng nguyn O157.Vic pht hin E.coli O157 bao gm 4 bc sau:- Tng sinh trong mi trng mTSB + N vi nhit 41,5C trong 6 gi, ri tip t 12 18 gi.

- Tch v lm giu E.coli O157 bng cc ht t min dch c bc khng khng nguyn c hiu ca E.coli O157.

- Phn lp chn lc trn mi trng thch cefixime tellurite sorbitol MacConkey (CT-SMAC) v mi trng phn lp chn lc th hai do la chn ca ngi s dng.

- Khng nh cc khun lc m tnh vi sorbitol trn mi trng CT-SMAC v cc khun lc in hnh ca E. coli O157 trn mi trng phn lp chn lc th hai bng phn ng sinh indol v phn ng kt dnh gia huyt thanh v khng huyt thanh ca E. coli O157. 3.2. Phm vi p dngTiu chun ny c th p dng sn phm thc phm v thc n chn nui.3.3. Ti liu vin dnISO 6887-1, Vi sinh vt trong thc phm v thc n chn nui - chun b mu th, dung dch huyn ph ban u v cc dung dch pha long thp phn kim tra vi sinh vt. Phn 1: cc nguyn tc chung chun b huyn ph ban u v cc dung dch pha long thp phn.ISO 7218, Vi sinh vt trong thc phm v thc n chn nui - Nguyn tc chung v kim tra vi sinh vt.3.4. Thit b, dng c, mi trng nui cy, thuc th v khng huyt thanh3.4.1 Thit b v dng cS dng cc thit b, dng c phng th nghim vi sinh c th nh sau:- T sy, ni hp p lc- T m, ni cch thy, my o pH- ng nghim, bnh cu, ng ong, pipet chia x ht, a petri, que cy- Thit b tch bng t tnh c gi t- Cc ht t tnh min dch khng Escherichia coli O157- ng nghim bng cht do kiu Eppendorf- B trn quay: c kh nng quay vi tc 15 vng/pht n 20 vng/pht- My trn vortex3.4.2. Mi trng nui cy v thuc th- Canh thang u tng trypton ci bin c novobioxin (mTSB + N)- Thch Macconkey sorbitol telurit xefixim (CT-SMAC)- Thch EMB hoc thch Endo- Thch dinh dng- Mi trng trypton/tryptophan- Thuc th Kovac- Dung dch mui 8,5%- Dung dch m phosphat ci bin 0,01 mol/l, pH 7,23.4.3. Khng huyt thanh Escherichia coli O1573.5. Chun b mu thChun b mu th theo cc tiu chun c th thch hp vi sn phm c lin quan. Nu khng c cc tiu chun c th ring lin quan n vic ly mu sn phm th cc bn lin quan nn tha thun.3.6. Cch tin hnh3.6.1. Quy trnh pht hin (xem ph lc 2)3.6.2. Thuyt minh quy trnh 3.6.2.1 . Phn mu th v huyn ph ban u chun b huyn ph ban u, cho x g hoc x ml phn mu th vo 9x ml hoc 9x g canh thang trypton ci bin c novobioxin (mTSB + N), c lm m s b trong t m 41,5oC thu c t l ca phn mu th v mTSB + N l 1/10 (khi lng/th tch hoc th tch/th tch).3.6.2.2. Tng sinh huyn ph ban u chun b theo 6.1 41,5oC/6 gi, ri tip 12 gi n 18 gi. Vic tch t min dch v cy ln a thch chn lc sau khi 6 gi c th cho cc kt qu dng tnh gi, sau khi tip 18 gi c th tr thnh m tnh.3.6.2.3. Tch bng t min dch (IMS)- Khi qut:

Sau khi t 12 gi n 18 gi th c sau 6 gi c th tin hnh IMS, nu cn. Cc ch dn di y l hng dn chung, v vy cn kt hp vi cc hng dn ca nh sn xut lin quan n cch tin hnh v phng php s dng cc b bt gi min dch v thit b cn thit.- Bt gi min dchVic s dng thit b tch bng t tnh v cc ht t min dch c ph khng th, cn thc hin quy trnh bt gi/tch di y:Trn dch cy tng sinh v yn cho nguyn liu th lng xung. Cho 20l cc ht t min dch c chun b nhit phng vo ng nghim Eppendorf. Ly 1 ml dch lng pha trn dch cy tng sinh, nu c th, trnh ht cc ht thc phm hoc cht bo, cho vo ng nghim Eppendorf.Trn huyn ph trn my trn quay vi tc khong t 12 vng/pht n 20 vng/pht trong 10 pht.Ch : S dng k thut v trng trnh mi nhim bn t bn ngoi v vic to bt kh. Thc hin trong t an ton v mang gng tay bo v.- Tcht tng ng Eppendorf vo gi t v cho cc ht t dnh kt da vo nam chm bng cch dch chuyn nh nhng gi t qua li 180o. M np cn thn m khng lm nhiu lon cc ht trn thnh ng. Dng pipet Pasteur v trng mi cho mi mu v mi ng nghim trong gi t, loi b dung dch bng cch ht t t dch lng t di y ng nghim. Thm 1ml dung dch m ra v trng v y np. Ly nam chm ra khi gi. Trn phn cha trong ng bng cch o nh nhng gi qua li 180o v t nam chm tr li gi.Ch : trnh lm nhim bn cho khi b sung dung dch m mi.Tin hnh nh trn, s dng pipet Pasteur v trng mi cho mi mu loi b dch ra. Lp li quy trnh ra vi ln. Ly ng nghim ra khi thit b t tnh, cho 100 l dung dch m ra v trng vo ng nghim v ha li cc ht t tnh.Ch : quy trnh ny c th gp phi kh khn i vi cc sn phm cht bo hoc phomat ti.3.6.2.4. Cy ln a thch chn lc v nhn dng cc khun lc E.coli O157 - Cy ln aS dng pipet man, ht 50 l cc ht t tnh ha li v ra vo a thch Macconkey sorbitol telurit xefixim lm kh v 50 l vo a mi trng phn lp th hai (do phng th nghim chn). Dng que cy vng v trng ria cy cc ht ny thu c nhiu khun lc tch bit r trn khp a thch. mi trng nui cy 37oC/18-24 gi v mi trng nui cy chn lc th hai nhit v thi gian theo quy nh.- Nhn dng cc khun lc E.coli O157 in hnhTrn a thch CT-SMAC: khun lc in hnh l nhng khun lc c ng knh xp x 1mm, trong sut, gn nh khng mu v vin ngoi c mu vng nu nht.Gii thch: vi khun E.coli O157 khng ln men c sorbitol nn khun lc khng mu. Trong khi cc dng vi khun E.coli khc ln men c sorbitol, sinh acid, pH mi trng chuyn sang acid nn cho khun lc mu hng.Trn a thch phn lp chn lc th hai: c im khun lc theo ch dn ca nh sn xut.

3.6.2.5. Khng nh- Chn khun lcLy 5 khun lc in hnh trn mi a, nu nh trn mi a c t hn 5 khun lc th ly tt c s khun lc c mt kim tra. Cy ria tng khun lc chn ln a thch dinh dng. mi trng nui cy 37oC/18-24 gi trong t m.- Khng nh bng tnh cht sinh vt ha hc: kh nng sinh indolChn mt khun lc thun khit trn a thch dinh dng cy vo ng ng mi trng trypton/tryptophan. mi trng nui cy 37oC/24 gi trong t m.Nh khong 1ml thuc th Kovac v yn 10 pht nhit phng.Kt qu: Xut hin vng mu trong ng mi trng nui cy chng t phn ng dng tnh, mu vng/nu chng t phn ng m tnh.Gii thch: Trong mi trng tryptophan, vi khun E.coli tit ra enzyme tryptophanase thy phn tryptophan thnh indol. Nhn bit indol bng thuc th Kovac nh sau: indol phn ng vi cht p-dimethylaminobenzaldehyde to thnh mui dimethyl ammonium c mu (phn ng dng tnh).- Khng nh bng huyt thanhCh kim tra cc khun lc dng tnh vi indol bng phn ng huyt thanh ca chng vi khng huyt thanh O157.+ Loi b cc vi khun phn lp t kt dnhCho 1 git nc NaCl 8,5% ln mt lam knh sch. Dng que cy v trng ly vi khun t a thch dinh dng, ha tan vi khun trong git nc NaCl 8,5% sao cho thu c huyn ph c v ng nht.Di chuyn phin knh nh nhng trong 30 giy n 60 giy. Quan st kt qu thu c da vo nn mu en v dng knh lp, nu cn.Kt qu: nu huyn ph to thnh cc mng c th nhn thy, th chng c coi l t kt dnh v khng c dng kim tra tip.+ Phn ng khng huyt thanh E.coli O157Dng que cy v trng ly vi khun t a thch dinh dng, ha tan vi khun trong git nc NaCl 8,5% sao cho thu c huyn ph c v ng nht. Thm mt git khng huyt thanh E.coli O157, trn u.Kt qu: nu trong vng 1 pht, thy xut hin hin tng dnh kt th c ngha l phn ng dng tnh, nu khng dnh kt th phn ng m tnh.+ Nhn dng dng tnhCc vi khun cho phn ng dng tnh vi indol v vi khng huyt thanh O157 hoc O157 cng vi khng huyt thanh H7 c coi l cc vi khun dng tnh.3.6.2.6. c trng khc nhn dng cc khun lc dng tnh cho vic pht hin cc khng nguyn roi v cc c tnh gy bnh, cn gi cc chng ny ti phng th nghim chun.3.7. m bo cht lng3.7.1. Kim tra cc chng v mc ch m bo cht lngCc chng E.coli O157 khng mang cc yu t c thuc loi gy bnh u c sn t c quan lu gi vi khun quc t hoc quc gia. Cc chng ny c dng kim tra v m bo cht lng mi trng v khng huyt thanh.3.7.2. Phng php cy kim tra kh nng ca phng th nghim v mi trng pht hin s lng nh E.coli O157 trong cc mu thc phm cn th nghim bng phng php m t trong tiu chun ny, th cc mu i chng c lng nh dch cy E.coli O157 khng gy bnh v lng ln dch cy ca chng E.coli khc cn c thc hin song song vi mu th.3.8. Biu th kt quTheo phn din gii cc kt qu, ghi li s c mt hay khng c mt Escherichia coli O157 trong phn mu th, tnh theo gam hoc theo mililit.3.9. Bo co th nghimBo co th nghim phi ghi r:- Mi thng tin cn thit nhn bit y v mu th- Phng php ly mu s dng, nu bit- Phng php th s dng, vin dn tiu chun ny- Nhit s dng

- Mi chi tit thao tc khng quy nh trong tiu chun ny, hoc ty la chn, cng vi cc chi tit bt thng khc c th nh hng ti kt qu.

- Cc kt qu th nghim thu c. Bo co kt qu cng phi nu r nu cc php th tip theo c thc hin bi phng th nghim chun, hoc nu thc hin th nu kt qu thu c.Ph lc 1MT S MI TRNG NUI CY3.1. Mi trng Macconkey sorbitol telurit xefixim (CT-SMAC)3.1.1. Mi trng c bn- Thnh phn:

Dch thy phn casein bng enzym: 17,0 gDch thy phn u tng bng enzym: 3,0 gSorbitol: 10,0 gMui mt: 1,5 gNaCl: 5,0 g trung tnh: 0,03 gTm tinh th: 0,001 gThch: 9g n 18 gNc: 1000 ml- Pha ch:Ha tan cc thnh phn c bn hoc mi trng hon chnh kh trong nc bng un si. Chnh pH sao cho sau khi kh trng pH l 7,1 0,2 25oC, nu cn. Kh trng trong ni hp p lc nhit 121oC/15 pht.3.1.2. Dung dch kali telurit- Thnh phn:Kali telurit: 0,25 gNc: 100 ml- Pha ch:Ha tan kali telurit trong nc v lc qua mng lc kh trng.Bo qun: 1 thng nhit 5oC 3oC, khi xut hin kt ta trng th loi b.3.1.3. Dung dch xefixim- Thnh phn:Xefixim: 5,0 mgNc: 100 ml- Pha ch:Ha tan xefixim trong nc v lc qua mng lc kh trng.Ch : Xefixim c th cn phi ha tan trong etanol.Dung dch ny bo qun c mt tun nhit 3oC 2oC.3.1.4. Mi trng hon chnh- Thnh phn:Mi trng c bn: 1000 mlDung dch kali telurit: 1,0 mlDung dch xefixim: 1,0 ml- Pha ch:

Lm ngui mi trng c bn kh trng n nhit khong t 44oC n 47oC, cho 1 ml dung dch kali telurit v 1 ml dung dch xefixim vo 1000 ml mi trng c bn.Nng cui cng ca kali telurit l 2,5 mg/l v xefixim l 0,05 mg/l.- Bo qun: mi trng ny c th bo qun c 2 tun nhit 3oC 2oC.3.2. Thch dinh dng- Thnh phn:Cao tht: 3,0 gPepton: 5,0 gThch: 9g n 18gNc: 1000 ml- Pha ch:Ha tan cc thnh phn hoc mi trng hon chnh kh trong nc bng cch un nng, nu cn. Chnh pH sao cho sau khi kh trng pH l 7,0 0,2 25OC, nu cn.Phn phi mi trng vo cc bnh c dung tch thch hp. Kh trng trong ni hp p lc 121oC/15 pht.- Bo qun:Mi trng ny c th bo qun c 2 tun nhit 3oC 2oC.3.3. Mi trng trypton/tryptophan- Thnh phn:Sn phm thy phn casein bng emym: 10 gDL-Tryptophan: 1 gNaCl: 10 gNc: 1000 ml- Pha ch:Ha tan cc thnh phn ny trong nc, un nng nu cn v lc. Chuyn cc lng mi trng 5 ml vo cc ng nghim. Kh trng 121oC/15 pht.3.4. Canh thang u tng trypton ci bin c novobioxin (mTSB+N)3.4.1. Canh thang u tng trypton ci bin- Thnh phn:Dch thy phn casein bng enzym: 17 gDch thy phn u tng bng enzym: 3 gD(+)-glucose: 2,5 gMui mt: 1,5 gNaCl: 5,0 gK2HPO4: 4,0 gNc: 1000 ml- Pha ch:Ha tan cc thnh phn trn hoc mi trng hon chnh kh trong nc, bng cch un nng, nu cn. Chnh pH sao cho sau khi kh trng pH= 7,4 0,2 25oC, nu cn.Phn phi mi trng vo cc bnh c dung tch thch hp. Kh trng trong ni hp p lc 121oC/15 pht.3.4.2. Dung dch novobioxin- Thnh phn:Novobioxin: 0,45 gNc: 100 ml- Pha ch:Ha tan novobioxin trong nc v lc qua mng lc kh trng.3.4.3. Mi trng hon chnhNgay trc khi s dng, cho 1ml hoc 4 ml dung dc novobioxin vo 225 hoc 900 ml mi trng mTSB ngui.Nng cui cng ca mTSB l 20mg/l.3.5. Thuc th kovacs- Thnh phn: 4-dimethylaminbenzaldehyd: 5g2-metylbutan-1-ol hoc pentan: 75mlAcid clohydric 1.18 n 1.19 g/ml: 25ml- Pha ch: Ha tan 4-demethylaminbenzaldehyd trong cn bng cch un nng nh trong ni cch thy duy tr nhit 50 - 55oC, lm ngui v thm acid.- Bo qun: trnh nh sng , nhit 40oC.Thuc th phi c mu vng nht hoc nu nht.3.6. Dung dch m ra (dung dch m phosphat ci bin 0,01mol/l - pH 7,2)- Thnh phn:NaCl: 8 gKCl: 0,2 gNa2HPO4 (khan): 1,44 gKH2PO4 (khan): 0,24 gPolyoxyetylen sorbitan monolaurat: 0,2 mlNc: 1000 ml- Pha ch:Ha tan cc thnh phn trong nc, chnh pH n 7,2 0,2 25oC, nu cn. Phn phi mi trng vo cc bnh cu c dung tch thch hp. Kh trng trong ni hp p lc 121oC/15 pht. Ph lc 2

QUY TRNH PHT HIN VI KHUN E.COLI O157 THEO TIU CHUN ISO 16654:2001

Hnh 3.1. Khun lc E. coli O157 (khng mu, khng ln men sorbitol) trn CT-SMAC

Hnh 3.2. Khun lc E. coli khng phi O157 (mu hng, ln men sorbitol) trn CT-SMAC

Ly mu kim tra (x gam hoc x mililit)

9x (ml) mTSB + N, lm m s b n 41,5oC

trong 6 gi ri lc u v tip t 12 18 gi 41,50C

Tch E.coli O157 bng cc ht t min dch v ra vi m v trng

41,50C

Ha tan cc ht t trong 0,1 ml m v trng

41,50C

50 l dch ht t c ha tan trn mi trng chn lc thu khun lc

41,50C

Mi trng CT-SMAC

41,50C

Mi trng phn lp th hai

41,50C

khng nh y l E.coli O157, cn xc nh thm kh nng sinh indole v phn ng gia huyt thanh vi khng khng nguyn ca E.coli O157

41,50C

Chn 5 khun lc c trng

41,50C

nhit v thi gian yu cu ca nh sn xut

Chn 5 khun lc c trng khng ln men sorbitol

41,50C

37oC trong 18 24 gi

41,50C

+

14