Quy trnh nh tnh Vibrio cholerae

Quy trnh nh tnh Vibrio cholerae

Danh sch nhm:H v tnM s sinh vinLp

H Th M Hnh200512019603DHTP5

L Th Huyn200512034703DHTP5

Nguyn Thanh Huyn202212023903DHDB3

Nguyn Th M Hng202212015703DHDB2

Trn Th Hng200512020703DHTP5

Thiu Th Tho Linh200512019603DHTP2

Hunh Nguyn Quang Tuyn03DHTP2

MC LC1. Tnh hnh ng c thc phm do vi khun Vibrio cholerae41.1.Tnh hnh dch t trn th gii41.2.Tnh hnh t ti Vit Nam52.c im vi khun Vibrio cholerae53.Cc phng php phn tch vi khun Vibrio cholerae73.1.Phng php truyn thng73.2.Phng php phn tch hin i83.2.1Phng php min dch hc ELISA (Enzyme Linked mmunosorbent Assays)83.2.2.K thut PCR chun (Polymerase Chain Reaction)93.2.3.Phng phap lai phn t (Hybridization)104.Quy trnh nh tnh Vibrio cholera114.1.Phm vi p dng114.2.Nguyn tc114.3.Thit b v dng c114.4.Mi trng v ha cht, mc ch s dng114.5.Quy trnh phn tch134.6.Cc bc tin hnh144.7.c kt qu21Ti liu tham kho22

Li m uVibrio cholerae (cn gi l Kommabacillus) l mt loi vi trng gram m gy bnh t ngi. Vibrio cholerae c nh gii phu hc ngi Filippo Pacini xc nh gy ra bnh t vo nm 1854, tuy vy khm ph ca ng khng c cng nhn rng ri n khi Robert Koch nghin cu c lp sau ba mi nm v cng b thng tin v bnh cng nh phng php phng chng.Bnh t hu ht c lin quan n thc phm. Mt nghin cu bnh chng ca Vin V sinh dch t Trung ng cho thy cc yu t nguy c cao mc t l: n tht ch, mm tm, rau sng, tit canh. Bnh ly theo ng tiu ho thng qua ngun nc, thc phm, rau qu... c bit l mt s hi sn nh s, c, hn c bt t nhng ni nhim hoc tay bn, dng c n ung b nhim, qua rui, nhng, chut, dn... lm ly lan mm bnh.Hng nm trn th gii c khong 3-5 triu ca mc t, 100.000-120.000 trng hp t vong. V vy, vic tm hiu quy trnh, phng php phn tch vi khun Vibrio cholerae l rt cn thit bo v sc khe cho ngi tiu dng v cng ng, Vibrio cholerae c th c pht hin thng qua nhiu phng php truyn thng hoc phng php min dch v sinh hc phn t khc nhau.

1. Tnh hnh ng c thc phm do vi khun Vibrio cholerae1.1. Tnh hnh dch t trn th giiBnh t c ni n t thi Hippocrates v Sanskrit, c m t ln u tin bi Garcia del Huerto nm 1563 v c John Snow chng minh c vai tr ly truyn ca nc nm 1849. Trong vng gn 200 nm qua loi ngi tri qua qua 7 v i dch t: i dch t ln th nht (1816-1826): Bt u t Bengal sau lan sang n , Trung Quc v bin Caspian. i dch t ln th hai (1829-1851): Nm 1831 dch lan sang London (6.536 ngi cht), Paris (20.000 ngi cht trong tng s 650.000 dn), tng s cht ton nc Php l 100.000. Sau dch lan sang Lin X, Quebec, Ontario v New York i dch t ln th ba (1852-1860): Xy ra nhiu vng ca Lin X, lm hng triu ngi cht. London, dch lm 10.738 ngi cht. Dch xy ra Chicago lm cht 5,5% dn s. i dch t ln th bn (1863-1875): Dch xy ra ch yu Chu u v Chu Phi. Dch xy ra London lm cht 5.596 ngi. i dch t ln th nm (1881-1896): Nm 1892 dch xy ra Hamburg lm 8.600 ngi cht. y l v dch nng cui cng Chu u. i dch t ln th su (1899-1923): Dch Chu u gim i do cc iu kin v sinh tt hn nhng dch vn xy ra nng n cc thnh ph ca Lin X. i dch t ln th by (1961 - nhng nm 70): Dch bt u Indonesia nm 1963, vi chng vi khun El Tor, sau lan sang Bangladesh (1963), n (1964), Lin X (1966). T Bc Phi dch lan sang Italy nm 1973, Nht Bn v Nam Thi Bnh Dng. T 1/1991 - 9/1994, dch xy ra Nam M, bt u Peru vi 1 triu ngi mc v 10.000 ngi cht, do chng t O1 - El Tor gy nn, c khc mt cht vi chng t ca i dch ln th by l s ti t hp gia chng t c in v t El Tor. T n nay, dch thng xuyn xy ra mt nc Chu Phi, Chu v M La Tinh. T vn l mt e do ton cu v y t cng cng v l mt trong cc ch s v pht trin x hi. Nm 2006, s ca t bo co trn th gii tng ln ng k, tng 79% so vi nm 2005 vi tng s l 236.896 c bo co t 52 nc, 6.311 trng hp t vong. Theo T chc Y t Th gii con s ny ch chim 10% tng s ca bnh xy ra trn thc t.Nm 2014 dch t vn c din bin phc tp ti Trung M v ti cc quc gia Ty v Trung Phi.

1.2. Tnh hnh t ti Vit Nam Bnh t ln u tin xut hin Vit Nam nm 1850 vi 2 triu trng hp bnh c thng bo. T nm 1910-1938, hng nm s bnh nhn mc t c thng bo dao ng t 5.000 - 30.000 ngi. Bnh t El Tor ln u tin xut hin min Nam nm 1964 vi 20.009 ngi mc bnh trong 821 ngi t vong. T n nm 1975, min Trung v min Nam, bnh t xy ra di dng dch lu hnh. Hng nm c hng trm bnh nhn b bnh t c thng bo. Nm 1994, bnh t xut hin khu vc Ty Nguyn vi 1.459 bnh nhn . Sau nm 1975, do vic thng thng gia hai min Nam, Bc, bnh t ly lan ra min Bc v gy ra nhng v dch t ri rc Hi Phng. T nm 1993 -2004, dch xy ra c ba min Bc, Trung, Nam vi khong vi nghn ca bnh c bo co hng nm. Tuy nhin, bnh khng bng pht thnh dch ln, c rt t trng hp t vong. Cc nm 2005-2006, c nc khng ghi nhn trng hp no. T cui nm 2007, dch li bng pht 19 tnh/thnh ph pha Bc, hng ngn trng hp mc nhng khng c trng hp no t vong.Nm 2009 c 930.496 ca mc tiu chy, trong c 4 ngi cht. Nm 2008 c 853 ca bnh t, khng c ngi cht, nm 2009 c 479 ngi mc t, trong c mt ngi cht, nm 2010 c 606 ca bnh t, khng c ngi cht, v nm 2011 c ba ca mc t, khng c ngi cht.Cui thng 7/2014, ti huyn Bnh Chnh (TP H Ch Minh) bng pht mt dch tiu chy cp khin hn 10 ngi mc phi nhp vin iu tr , trong c 1 bnh nhi t vong. Kt qu xt nghim nui cy v PCR pht hin mu c bu dng tnh V.cholera O1 tup huyt thanh Inaba. Theo iu tra ngi bn c ly c ch u mi Bnh in gip ranh vi huyn Bnh Chnh.Vic pht hin vi khun t trong c bu vng c ngha ngun nc b nhim vi khun ny, c kh nng ly lan trong cng ng.2. c im vi khun Vibrio choleraeVibrio cholerae v cc loi khc thuc chi Vibrio thuc v lp gamma ca ngnh Proteobacteria. C hai chng Vibrio cholerae chnh, chng c in v chng El Tor, v mt s nhm huyt thanh (serogroup) khc.

Hnh 1: Vi khun Vibrio choleraeVibrio cholerae thuc h Vibrionaceae, l vi khun c hnh cong nh du phy, bt mu gram m, khng c v, khng sinh nha bo, di ng c nh c lng. Phy khun t d nui cy trong mi trng ngho dinh dng, pH kim (pH t 8,5-9,0) v mn.. mi trng thch hp nh trong nc, thc n, trong cc ng vt bin (c, cua, s bin...) v.v... nht l trong nhit lnh, phy khun t c th sng c vi ngy n 2-3 tun. D b dit bi nhit (800C/5 pht), bi ho cht thng thng v mi trng axit. Vibrio cholerae c mt trong nc, sa, thc phm, cn trng, thy sn, sinh c t rut v ni c t trong ng tiu ha. c t rut gn vo nim mc rut non, hot ho enzyme adenylcyclase dn n tng AMP vng, lm gim hp thu Na+, tng tit Cl- v nc gy tiu chy cp tnh. Phy khun t c th chuyn ha trong thin nhin, thay i tnh di truyn do t bin t chng khng gy dch c th thnh chng gy dch v khng nhiu loi khng sinh. L vi khun ph bin trong t nhin, gy dch t, dch bnh d pht sinh ni nc bn v thc phm b nhim trng.V. cholerae c khong 140 nhm huyt thanh v c chia thnh V. cholerae-O1 v V.cholerae non-O1(Vibrio cholerae khng ngng kt vi O1 cn c gi l chng NAG). V. cholerae O1 c phn thnh 2 typ sinh hc (biotyp) da theo c im kiu hnh l V. cholerae classica v V. cholerae eltor. Da vo c im cc quyt nh khng nguyn A, B, C ca khng nguyn thn O, V.cholerae c phn thnh 3 typ huyt thanh (serotyp) sau: Serotyp Ogawa (c quyt nh khng nguyn A, B). Serotyp Inaba (c quyt nh khng nguyn A, C). Serotyp Hikojima (c c ba quyt nh khng nguynA, B, C).V. cholerae-non O1 nhm huyt thanh O139 l th phm gy dch t n v mt s nc chu t nm 1992 n nay.Phy khun t gy bnh nh c t t (choleragen). y l ni c t c cu trc gm hai n nguyn: n nguyn A (trng lng phn t l 27 000 dalton, mang c tnh cao) v n nguyn B c tnh khng nguyn c hiu v mt cu ni A2 c tc dng kch thch tng AMP vng (Adenosin 3,5-cyclic mono phosphat3. Cc phng php phn tch vi khun Vibrio cholerae3.1. Phng php truyn thng Mi trng nui cy:Cc phng php truyn thng xc nh cc loi Vibrio trong thc phm theo tiu chun hin nay (ISO) cng c ph bin, ISO/TS 21872-1:2007 dng xc nh V. cholerae. Phng php truyn thng thng dng mi trng cha mui vi pH khon 8,6 nh mi trng tng sinh nc peptone kim (alkaline saline peptone water). Mi trng phn lp thng dng l TCBS, cc khun lc ca V.cholerae trn mi trng ny c c im l trn, bng v c mu vng. Tuy nhin nhng pht hin gn y cho thy mi trng TCBS c hn ch l khng pht hin c cc chng V.cholerae ln men saccharose chm dn n sai st trong pht hin. Mt mi trng c hiu hn cn quan tm l mi trng to mu (Chromogenic agar media) theo c ch enzyme nh chromID Vibrio agar (bioMrieux) hay CHROMagar Vibrio c th pht hin c chng ln men saccharose chm . Nguyn tcMt lng mu xc nh (10g hoc 25g) c tng sinh trong mi trng chn lc c trng. Cy phn lp t mi trng tng sinh sang mi trng phn bit chn lc c trng. Cy khun lc nghi ng trn mi trng phn lp c khng nh bng cc phn ng sinh ha v huyt thanh hc. Mi trng tng sinh chn lc cho V. cholerae l nc peptone kim. Mi trng thng dng phn lp Vibrio l TCBS (Thiosalphate Citrate Bile Sucrose Agar). Cc vi sinh vt len men c sucrose trong mi trng ny s cho khun lc mu vng v lm acid ha mi trng bn di khun lc. Nu vi sinh vt khng len men c ng sucrose s cho khun lc mu xanh.3.2. Phng php phn tch hin i3.2.1 Phng php min dch hc ELISA (Enzyme Linked mmunosorbent Assays) ELISA (Enzyme-Linked ImmunoSorbent Assay) hay EIA (Enzyme ImmunoAssay) l mt k thut sinh ha pht hin khng th hay khng nguyn trong mu xt nghim. K thut ELISA c th c s dng pht hin bnh pht sng do V.harveyi trn tm, mu nc v c th xc nh tt c cc dng Vibrio spp. Phng php ny c thc hin da trn nguyn tc bt dnh c hiu ca khng nguyn v khng th v gm cc bc c bn sau: Khng nguyn cha bit c gn trn mt b mt. Khng th bit trc c "ra" qua b mt . Khng th ny c gn kt vi enzyme. Thm vo mt c cht (substance): enzyme s bin i c cht ny v to tn hiu c th xc nh c. i vi cc ELISA pht quang, nh sng s c pht ra t mu cha khng nguyn khng th. S hin din ca phc hp khng nguyn khng th s quyt nh cng sng pht ra. Vi nguyn l trn, ELISA gip xc nh s c mt hay khng c mt cng nh lng khng nguyn trong mu nghin cu. tin hnh ELISA cn phi c t nht mt khng th c hiu cho khng nguyn cha bit. Thng thng khng nguyn c c nh ti cc ging ca vi phim (polystyrene microtiter plate). Phng thc c nh khng c hiu: khng nguyn gn trc tip vo b mt ca a. Phng thc gn c hiu ("sandwich" ELISA):khng nguyn c gn vi mt khng th c hiu cho cng khng nguyn cn kim tra. Khng th c hiu s c thm vo, phn ng to phc hp khng nguyn khng th c th sy ra. Nu khng th c gn trc tip vi enzyme, tn hiu quang hc do enzyme lm bin i c cht s gip pht hin khng nguyn cn kim tra. Trong trng hp s dng khng th th cp (secondary antibody) c gn vi enzyme thng qua cc lin kt cng ha tr gia cc phn t sinh hc (bioconjugation), khng nguyn cn xc nh s c nhn bit qua khng th th cp ny. Gia cc bc ca ELISA, cc protein v cc khng th khng c hiu, khng th khng gn vi khng nguyn s c ly i nh cc loi dch c tc dng "ra". Sau bc "ra" cui cng, ch cn khng th lin kt vi khng nguyn c gi li. Sau khi c thm vo, c cht s chu tc dng ca enzyme lin kt vi khng th trong phc hp khng nguyn khng th. Phn ng pht quang (bin i c cht) s sy ra. Trc y cc c cht to mu sc c s dng trong ELISA nhng ngy nay cc cht pht quang c dng rng ri lm tng tnh c hiu v chnh xc ca ELISA. C nhng phng php ELISA nh sau: ELISA gin tip (indirect ELISA),Sandwich ELISA,ELISA cnh tranh (Competitive ELISA) Hnh 2: Phng php min dch hc ELISA3.2.2. K thut PCR chun (Polymerase Chain Reaction) Phng php PCR (Polymerase Chain Reaction) c Karl Mullis v cng s (M) pht minh nm 1985 v k t to nn mt tc ng to ln i vi cc nghin cu sinh hc trn ton th gii. y l phng php in vitro s dng cc cp mi tng hp s lng ln cc bn sao t mt trnh t DNA c bit da trn hot ng ca cc dNTP t do l enzyme polymerase, sau 20-35 chu k c th nhn c s b sao rt ln ca on DNA cn khuch i. K thut PCR chun ch thc hin c khi bit r cc trnh c hiu 2 u DNA cn nhn gen (on khun) c th thit k cc cp mi c hiu .Hin nay c th s dng k thut khuch i DNA chun on nhanh bnh do Vibrio spp trong vi gi m khng mt nhiu thi gian phn lp vi khun.Nguyn tc Nguyn tc ca phng php ny l pht hin mt on DNA c hiu cho Vibrio spp. Phng php PCR da trn hot ng ca DNA polymerase trong qu trnh tng hp DNA mi t mch khun. Tt c cc DNA polymerase u cn nhng mi, l nhng on DNA ngn c kh nng bt cp b sung vi mt u ca mch khun. on mi ny sau s c ni di ra nh hot ng ca DNA polymerase hnh thnh mt mch mi hon chnh. Trong mt phn ng PCR khuych i mt trnh t c bit no , ta cn phi c nhng thng tin ti thiu v trnh t thit k cc mi b sung hai u mch bao gm mt mi xui (sense primer) v mt mi ngc (antisense primer) so vi chiu phin m ca gene. Mt khi c cung cp hai mi b sung hai u, phn ng s cho ra hng lot bn sao ca on DNA nm gia hai mi .Cc giai on ca phn ng PCR Mt phn ng PCR l mt chui nhiu chu k ni tip nhau, mi chu k gm ba giai on: Giai on bin tnh: trong mt dung dch phn ng bao gm cc thnh phn cn thit cho s sao chp, phn t DNA c bin tnh nhit cao trong vng 30 giy 1pht, thng l 940C. Giai on bt cp: giai on ny, nhit c h thp cho php cc mi bt cp vi khun. Trong thc nghim, nhit ny dao ng trong khong t 30 70C khong 40 giy 1 pht. - Giai on tng hp: nhit c tng ln n 720C cho DNA polymerase s dng vn l polymerase chu nhit hot ng tng hp tt nht. Thi gian ty thuc di ca trnh t DNA cn khuch i, thng ko di t 20 giy n nhiu pht. Trong phn ng PCR, chu k ba giai on s c lp li nhiu ln, mi ln lm tng s lng bn sao ln gp i. S khuch i ca phn ng l theo cp s nhn v theo tnh ton, sau 30 chu k, s khuch i s l 106 so vi s lng bn mu ban u. Sau phan ng PCR, cac DNA c nhum bi ethidium bromide va co th quan sat thy thng qua in di san phm trong agarose va quan sat di tia UV. 3.2.3. Phng phap lai phn t (Hybridization)Phng phap nay s dung mu do phat hin vi sinh vt trong thc phm da trn s phat hin mt oan gen c trng cua vi sinh vt. C s cua vic s dung mu do la lai phn t. Qua trinh bao gm s tach ri hai mach i cua chui xoan kep DNAkhi nhit vt qua nhit nong chay cua DNA (T m) va s tai bt cp cua cac oan DNA co trinh t nucleotide b sung khi nhit tr lai binh thng. Mt trong 2 mach DNA b sung c c inh trn gia th rn va nm nm ngay trn t bao hay m. S lai phn t say ra khi oan mi co trinh t nucleotide b sung vi mt vung trinh t trn DNA muc tiu gp nhau do chuyn ng cua nhit va khi nhit mi trng thp hn T m it nht vai .4. Quy trnh nh tnh Vibrio cholera4.1. Phm vi p dngPhng php ny tham chiu theo TCVN 7905-1:2008 (ISO 21872-1:2007) dng pht hin Vibrio cholerae trong thc phm v thc n chn nui.4.2. Nguyn tcPht hin V.cholerae trong thc phm c thc hin bng cch cn mt lng mu xc nh v nui trong mi trng lng chn lc. T y dch khun c cy chuyn sang mi trng rn chn lc. nhng khun lc ging V.cholerae s c th nghim cc phn ng sinh ha.Yu cu chungVic pht hin Vibrio cholerae cn n bn giai on lin tip .

4.3. Thit b v dng c 5. 6. Thit b v dng c thy tinh6. CH THCH: C th dng dng c s dng mt ln thay th cho cc dng c thy tinh s dng nhiu ln nu p ng c cc yu cu tng t.7. S dng cc thit b, dng c ca phng th nghim vi sinh thng thng [xem TCVN 6404 (ISO 7218)] v c th nh sau:8. 6.1. T m, c th duy tr nhit 37 0C 1 0C.9. 6.2. T m hoc ni cch thy, c th duy tr nhit 41,5 0C 1 0C.10. 6.3. Ni cch thy, c th duy tr nhit t 44 0C n 47 0C.11. 6.4. Ni cch thy, c th duy tr nhit 37 0C 1 0C.12. Nn s dng cc ni cch thy (6.2, 6.3 v 6.4) c cha cht khng khun.13. 7. Ly mu14. iu quan trng l mu gi n phng th nghim phi ng l mu i din v khng b h hng hoc b thay i trong sut qu trnh vn chuyn v bo qun.15. Vic ly mu khng quy nh tiu chun ny. Nn ly mu theo tiu chun c th lin quan n sn phm. Nu khng c tiu chun ring v ly mu sn phm c lin quan th cc bn t tha thun v vn ny.16. 8. Chun b mu th17. Chun b mu th theo phn tng ng ca TCVN 6507 (ISO 6887) v/hoc TCVN 6263 (ISO 8261) v tiu chun c lin quan n sn phm. Nu khng tiu chun ring th cc bn t tha thun v vn ny.

17.1. Mi trng v ha cht, mc ch s dngBng 1: Mi trng v ha cht, mc ch s dngMi trng v ha chtMc ch

APWTng sinh chn lc: phc hi sc sng ca cc vi sinh vt b tn thng v nhm gia tng tng mt tng i ca vi sinh vt cn pht hin, c ch s pht trin ca cc vi sinh vt khng mong mun.

TCBS (Thiosulphate citrate bile salt sucrose)Phn lp: Mi trng thch Thiosulfate Citrate Bile Salts Sucrose l mi trng chn lc dng phn lp cc loi Vibrio t mu xt nghim phn c ln vi vi khun khc. Cc loi Vibrio c phn bit bng c im do khun lc sinh ra trn mi trng. Cc kiu khun lc khc nhau c sinh ra t cc loi Vibrio khc nhau, th hin qua s ln men ca sucrose. Khi dng thch TCBS, chng ta nn dng cht nui cy c, nhng loi Vibrio d cht t ngt v mi trng ny c ngn chn c iu . Mu xt nghim ti l tt nht, v vi sinh vt ny nhy cm vi iu kin kh, nh sng v pH acid

NA/TSAPhc hi

TSI/KIAKhng inh s b: Mi trng thch Kligler Iron Agar c s dng nui cy trc tip chotrc khun gram m ln men ng glucose, y l c im c bn trong phn loi bc u ca cc trc khun gram m. Mi trng ny cng ng thi th tnh ln men ng lactose, sinh hi trong qu trnh ln men carbohydrat v sinh H2S. Tt c nhng c im c s dng cho cc trc khun gram m thuc h vi khun ng rut

a giy OxidaseKhng nh s b: dng th nghim Oxidase

Dung dch NaClKhng nh s b: mi trng th nghim tnh chu mn

KOH 3%Th Strinrg test khng nh s b

Arginine dehydrolaseTh nghim sinh ha khng nh

Ornithine decarboxylase

Lysine decarboxylase

TW (Tryptone Water)

ONPG

KowacsThuc th Indol

HCl v NaOH (KOH) 10%Chnh pH

Khng huyt thanh polyvalent OTh nghim khng huyt thanh

17.2. Quy trnh phn tch6-8 ging nht 25g mu vi 225 ASPWTng sinh ln 1Tng sinh ln 2Phn lp ln TCBS 37.0 0.5 0C trong khong 24 giV. cholerae : Khun lc vng, ng knh 3-4mmCy chuyn sang BHI hay TSA c 1% NaClSinh ha s b, khng nh, khng huyt thanhKT LUN

17.3. Cc bc tin hnh Bc 1: Chun b mu Cn chnh xc 25g (10g) mu cho vo bao PE v trng, sau thm 225ml (90ml) dung dch pha long mu APW. Tin hnh ng nht mu bng my dp mu. Thi gian dp mu l 60 giy. Tt c cc thao tc phi thc hin trong iu kin v trng. Bc 2: Tng sinh chn lc Dng nc peptone kim cha 1% mui NaCl sau em trong 24 gi. Vibrio cholerae c th c mt mt lng nh v thng i km vi mt lng ln ng k cc loi vi sinh vt khc thuc h Vibrionaceae hoc cc h khc. Do , cn phi c hai bc tng sinh chn lc lin tip pht hin cc vi sinh vt ch ny. Tng sinh ln u trong mi trng lng chn lcCy phn mu th vo mi trng tng sinh nhit mi trng. huyn ph ban u 37 0C trong 6 h 1 h i vi cc sn phm ng lnh su hoc 41,5 0C trong 6 h 1 h i vi sn phm ti, kh hoc sn phm p mui.Cn ch khi p dng ton b phng php cho cc sn phm c hm lng mui cao, v nng mui cui cng trong mi trng c th lm thay i cc c tnh Tng sinh ln th hai trong mi trng lng chn lcChuyn 1 ml dch cy thu c trong 9.2 c ly t b mt cho vo ng nghim cha 10 ml mi trng APW. trong mi trng APW trong 18 h 1 h nhit 41,5 0C. Bc 3: Phn lp Sau 24 gi, dng que cy vng ly mt t sinh khi trn mi trng APW v cy ln b mt a mi trng thch TCBS phn lp khun lc n, 37oC trong 24 gi. Trn mi trng thch TCBS khun lc V. cholerae trn, ln c mu vng, ng knh 2-3mm.

Hnh 3: Khun lc V.cholerae trn mi trng TCBS

Bc 4: Phc hi trn mi trng dinh dng NA/TSAnh du 5 khun lc in hnh hoc nghi ng t a trn mi trng phn lp TCBS. Nu trn mt a c t hn nm khun lc in hnh hoc khun lc nghi ng, th ly tt c cc khun lc in hnh hoc nghi ng cy ria ln NA/TSA c b sung 1,5% NaCl. 3710C trong 24 gi. Trng hp 1: t mi a th mt khun lc c trng, nu cho cc kt qu th nghim sinh ha ph hp th kt lun pht hin. Trng hp 2: nu khun lc u tin cho kt qu sinh ha khng ph hp th tin hnh th bn khun lc cn li c nh du, mt trong bn khun lc ny cho kt qu th nghim sinh ha ph hp th kt lun pht hin v ngc li th kt lun khng pht hin.Cc khun lc chn cy ria ln NA/TSA c s dng cho cc th nghim sinh ha. Bc 5: Khng nh sinh ha v khng huyt thanh khng nh, t mi trng chn lc, ly ra t nht nm khun lc c coi l in hnh hoc ging vi tng V. cholease c kh nng gy bnh cy truyn. Nu trn a c t hn nm khun lc th cy truyn tt c cc khun lc ny.Cc loi thc phm, c bit l hi sn c th cha mt lng ln vi khun, k c Vibrio spp. khng gy bnh m c th pht hin nh qu trnh cy chn lc. Vic cy truyn cc lng nh cc khun lc c th lm tht thot cc loi c kh nng gy bnh.Th nghim sinh ha : Bc 5.1. Th nghim s bBng 2: Cc php th nghim s bPhp thPhn ng tiu biu ca V. cholera

Tnh chu mn 0% NaCl 2% 6% 8% 10%++---

Tnh di ng+

Oxidase+

Th nghim vi cc canh thang trypton mui Cy khun lc t mi trng TSA vo cc ng canh thang mui trypton c gii nng NaCl l: 0; 1; 2; 6; 8 v 10 %. Nui nhit 37oC trong 18 - 24 gi, ri li cc ng m tnh sau 18 - 24 gi na. V. cholerae pht trin c trong ng canh thang c nng NaCl l 0%, 2% v lm c mi trng. Th nghim tnh di ng Nguyn tc: Xc nh kh nng di ng ca V. cholerae Dng que cy thng ly vi khun sang ng nghim thch bn lng NA, m thng t trn xung di ngay gia ng mi trng, 370C trong 24 gi. Phn ng dng tnh khi V. cholerae lan ra khi ng cy, xa hay gn ty theo kh nng di ng ca vi khun mnh hay yu. Phn ng m tnh khi V. cholerae ch mc trn ng cy.

Hnh 4: Th nghim tnh di ng Th nghim oxidaza Nguyn tc: Xc nh s hin din ca h enzyme oxidase V. cholera.t giy lc ln mt np a petri, nh khong 2 - 3 git thuc th oxidaza. Dng que cy bch kim (platin) hoc que cy nha dng mt ln; hoc que g v trng ly cc khun lc t mi trng TSA ria ln v tr nh thuc th oxidaza. V. cholerae lm vt ria c mu tm thm hoc xanh thm sau 30 giy n 1 pht. Khng dng que cy bng crm hoc bng st trong th nghim ny.Bc 5.2. Th nghim khng nhBng 3: Cc php th nghim khng nhPhp thPhn ng tiu biu ca V. cholera

ADH-

ODC+

LDC+

ONPG+

TSISinh kh (glucoza)-

Lactoza-

Sacaroza+

Indol+

Th nghim decarboxylase+ADH: enzyme arginine dihydrolase+ODC: enzyme ornithine decarboxylase+LDC: enzyme lysine decarboxylaseCy vi khun t mi trng TSA vo cc ng canh thang ornithine, lysine, arginine v ng i chng khng c axit amin. Mi ng c ph 1-2 ml du khong v trng. cc ng nhit 370C trong 24 gi. Phn ng dng tnh khi mi trng tr nn c v c mu tm. Phn ng m tnh khi ton b ng canh thang c mu vng n nh trong 4 ngy ( tng t mu ca ng i chng).V. cholera cho phn ng lysine v ornithine dng tnh, phn ng arginine m tnh.

Hnh 5: Th nghim decarboxylase Th nghim ONPG:Xc nh s hin din ca enzyme -galactosidase vi sinh vt. Cc vi khun ch ln men lactose khi c s tng hp trong t bo ca hai ezyme l -galactosidase c vai tr xc tc s thy phn lactose v permease c vai tr vn chuyn lactose vo bn trong t bo.Ly vi khun pht trin trn mi trng TSI hoc t mi trng khng chn lc khc c cha 1,0% lactose vo ng nghim c cha 0,25 ml nc mui sinh l, ha tan. Thm 1 git toluene vo ng ri lc u, yn trong 5 pht. Thm 0,25 ml dung dch ONPG, ng nghim nhit 370C. V. cholerae cho phn ng ONPG dng tnh khi mi trng chuyn sang vng, m tnh khi mi trng khng chuyn mu.

Hnh 6: Th nghim ONPG Th nghim trn mi trng TSI, KIA(Kligler Iron Agar):+ Mi trng KIA gm glucose v lactose (c th ln men carbohydrat), phenol (ch th pH), pepton (ngun carbon/nitrogen) v mui st cng vi thiosulfate natri.+ Mi trng TSI gm Pepton, dch chit tht, dch chit nm men, NaCl, Lactose, Sacarose, Glucose, st (III) xitrat, phenol, thch, nc.Nguyn tc: nhm xc nh vi sinh vt s dng cc ngun carbohydrate no, c sinh ga hay khng, c sinh H2S hay khng. Cy chuyn cc khun lc t mi trng TSA vo cc ng thch nghing TSI hoc KIA. Ni lng cc np ng to iu kin hiu kh. Nui nhit 37oC trong 18 - 24 gi. Trn mi trng TSI, V.cholerae lm phn thch nghing v thch ng ca mi trng chuyn mu vng, khng sinh hi v khng sinh H2S. Trn mi trng KIA, phn thch nghing c mu v thch ng mu vng, khng sinh hi v khng sinh H2S, V. cholerae ln men ng glucose nhng khng ln men ng lactose.Thi gian khng qu 24 h, v cy b mt nghing th mu vng V. cholerae c th chuyn sang mu sau 24 h.

Hnh 7: Th nghim KIA/TSI

Th nghim indolCy khun nghi ng vo ng cha 5 ml mi trng mui tryton-tryptophan (5.10). 37 0C trong 24 h-3 h. Sau khi , thm 1 ml thuc th Kowacs.Vic hnh thnh vng mu phn ng dng tnh (hnh thnh indol). Vng mu nu-vng chng t phn ng m tnh.Th nghim khng huyt thanhNh 1 git khng huyt thanh ln lam knh sch v mt git nc mui sinh l ln mt v tr khc ca lam. Dng que cy v trng khc nhau chuyn vi khun t TSA ln hai git dung dch trn ri phn tn u vi khun. Kt lun dng tnh khi c hin tng ngng kt git c khng huyt thanh v khng c hin tng ngng kt git nc mui. S dng chng chng dng v chng m trong qu trnh thc hin.Bng 4: Phn bit V.cholerae O1 v V.cholerae non-O1Khng nguynKhng huyt thanh a gi O1Dung dch mui sinh lKt lun

Chng c kt qu sinh ha tng t V.cholerae+-V.cholerae O1

Chng c kt qu sinh ha tng t V.cholerae--V.cholerae non-O1

Chng c kt qu sinh ha tng t V.cholerae++Chng khng c hiu vi khng nguyn O nhm 1

Bng 5: Phn bit cc kiu huyt thanh chng V. cholera O1Khng nguynKhng huyt InabaKhng huyt OgawaDung dch mui sinh lKt lun

V. cholera O1+--Chng Inaba

V. cholera O1-+-Chng Ogawa

V. cholera O1++-Chng Hikojima

V. cholera O1---Chng khng c hiu

17.4. c kt qu Pht hin hoc khng pht hin c V. cholerae trong 25 g mu rn hoc 25 ml mu lng.

Ti liu tham kho[1] Bi ging phn tch vi sinh thc phm, Trng i hc Cng nghip thc phm TP.HCM,2014.[2] Trn Linh Thc(chu bin), Phng phap phn tich vi sinh vt trong nc, thc phm va mi phm, NXB Giao Duc Vit Nam.[3]Phn tch vi sinh thc phm, Trng i hc Cng nghip thc phm TP.HCM, Nguyn Thy Hng, 2011.[4]http://www.dieutri.vn/bgtruyennhiem/2-11-2012/S2975/Bai-giang-benh-ta-Cholera.htm#ixzz3D127RjvE[5]http://www.case.vn/vi-VN/34/96/114/details.case[6]http://doc.edu.vn/tai-lieu/khoa-luan-tim-hieu-quy-trinh-phat-hien-vibrio-trong-thuy-hai-san-dong-lanh-52639/[7] TCVN 7905-1:2008, ISO/TS 21872-1:2007, vi sinh vt trong thc phm v thc n chn nui phng php pht hin Vibrio spp. C kh nng gy bnh ng rut phn 1: pht hin Vibrio parahaemolyticus v Vibrio cholerae[8]http://www.nihe.org.vn/new-vn/dao-tao-ngan-han--tap-huan-412312272/511/Dac-tinh-mot-so-moi-truong-phan-lap-va-chan-doan-vi-khuan-gay-benh.vhtmGVHD: Nguyn Th Kim OanhPage 13