1. H thng qun l da trn tiu chun (ISO9000, ISO14000,

HACCP. ... phi hp lin ngnh cng tc kim tra, thanh tra cht lng, v sinh an ton thc phm, ... 2.3. HACCP c gii thiu trong tiu chun ca CODEX mang s

hiu CAC/RCP 1-1969, Rev.4-2003, v tiu chun quc gia ca Vit Nam tng ng l TCVN 5603:2008. H THNG QUN L CHT LNG V SINH AN TON THC PHM THEO GMP V HACCP I. S ra i v ngha ca GMP v HACCP GMP (Good Manufacturing Practice) a ra cc ni dung c bn ca iu kin thc hnh sn sut tt, nhm kim sot tt c cc yu t nh hng ti an ton v sinh thc phm trong qu trnh ch bin thc phm t thit k, xy lp nh xng, thit b, dng c ch bin, iu kin phc v cho cng vic chun b ch bin n qu trnh ch bin, bao gi, bo qun v con ngi thc hin cc thao tc ch bin thc phm. Nhng qui nh ny to iu kin cho cc c s sn xut thc phm xy dng nhng qui phm c th ph hp vi iu kin, qui m, trnh cng ngh ca mnh. Cc qui nh a ra cc nguyn tc thc hnh v sinh chung p dng trong ch bin thc phm (bao gm nui trng v thu hi, x l, ch bin, ng gi, lu gi, vn chuyn, phn phi v bn) cho ngi m bo thc phm sn xut ra an ton, lnh mnh v b dng. Ngoi ra n cn nhm mc ch, da trn c s xy dng cc qui phm v thc hnh v sinh thc phm cho cc hng ho hay cc nhm hng c bit i hi phi p ng cc yu cu c bit v v sinh thc phm. HACCP c hnh thnh vo nhng nm 1960 khi cng ty Pillsbury ca qun i M v c quan nghin cu hng khng M (NASA) cng phi hp tm cch sn xut cc thc phm an ton cho cc chng trnh khng gian. NASA mun c mt chng trnh hon ton khng khuyt tt m bo an ton cho cc thc phm m cc nh du hnh dng trong v tr. Cng ty Pillsbury xut v ci bin HACCP thnh h thng c th to mc an ton cao nht song vn c th gim nh hot ng ly mu v th

nghim khu thnh phm. HACCP ch trng vo vic kim sot ti cc cng on v dng cc k thut gim st thng xuyn ti cc Im kim sot trng yu ngay t cc bc u tin trong qu trnh ch bin. Nhng khi nim v HACCP c Pillsbury trnh by cng khai ti hi ngh bo v thc phm nm 1971. Nm 1974 nhng nguyn tc ca HACCP c c quan Thc phm v dc phm M (FDA) thc hin y i vi cc loi thc phm ng hp. Vo nhng nm 80 phng thc ny c nhiu cng ty thc phm c ting khc trin khai p dng. Nm 1985, Vin hn lm Khoa hc Quc gia M khuyn co rng m bo an ton cho thc phm, cc qu trnh ch bin chng phi tun theo cc yu cu ca h thng HACCP. Gn y nhiu t chc Quc t nh U ban quc t v thc phm( ICMSF), Hip hi quc t v sa, thc phm v v sinh mi trng (IAMFES) c nhiu khuyn co v p dng HACCP. Phng php HACCP mang tnh h thng v khoa hc, n xc nh cc mi nguy hi c bit cng nh cc gii php kim sot nhm m bo tnh an ton cho thc phm. N l cng c nh gi cc mi nguy hi v xc lp cc h thng kim sot ch trng vo vic ngn nga hn l vic th nghim ti khu thnh phm. Trong mi trng hp, h thng HACCP u to mi iu kin cho s pht trin cng ngh v quy trnh ch bin tin b. II. Ni dung ca GMP v HACCP Cc yu cu ca GMP: - Yu cu v nh, xng v phng tin ch bin: Yu cu ny i hi khi xy dng c s ch bin thc phm cn phi xem xt n v tr sao cho ph hp vi mt c s ch bin thc phm phi sch s thong mt, khng gy nhim mi trng, khng t

nhng ni c mi trng khng lnh mnh .v.v. - Qui nh v cc yu cu kim sot v sinh nh xng nh: X l nc thi, sn phm ph, bo qun ho cht gy him, kim sot sinh vt gy hi v dng cc nhn - Kim sot qu trnh ch bin : Kim tra tt c mi hot ng phi thc hin theo nguyn tc v sinh c bn ca GMP, phi c cc bin php kim sot cht lng sao cho cc im kim sot quan trng c kim sot trong sut qu trnh ch bin. Thc hin cc bin php phng sn phm b nhim bn, th cc ch tiu vi sinh ho hc, tp cht khu cn thit xc nh nguy c gy nhim. Cc sn phm b nhim bn hay bin cht phi b loi b hoc c x l gim bt c cht. - Yu cu v con ngi: i vi c s ch bin thc phm th yu cu i vi con ngi khi tuyn vo lm vic l ht sc quan trng. Nht thit phi kim tra sc khe ( v th lc, tr lc v bnh tt) ca tt c mi ngi , c bit vi nhng cng nhn tip xc trc tip vi thc phm trnh ly bnh truyn nhim. Phi a ra nhng qui nh trong vic khm sc kho cho cn b cng nhn vin nh khm nh k m bo ch c nhng ngi c tiu chun sc kho mi c tip tc lm vic trong c s sn xut thc phm .Phi thng xuyn gio dc cho cc cn b cng nhn vin trong c s mnh bit gi gn sc kho v v sinh cc nhn m bo cc yu cu ra. - Kim sot khu bo qun v phn phi: a ra cc yu cu v vic vn chuyn v bo qun sao cho thnh phm phi m bo trnh nhim bn thc phm bi cc tc nhn vt l, ho hc, vi sinh. . . v khng lm phn hu thc phm. Qua nhng yu cu nu trn cho thy, GMP c c th coi l h thng tin , n cp n tt c mi yu t ti thiu c lin quan ti cht lng v sinh trong ch bin thc phm. T v tr ca c s n cu trc ca tng b phn, t dy chuyn cng ngh ti mi trng, t thit b, dng c ti con ngi, t cht ph thi ti sn phm trung gian, ph gia, nguyn vt liu v thnh phm. Xy dng thnh cng GMP s m bo cho doanh nghip chc chn c sn phm t cht lng cao v v sinh an ton thc phm. III. Cc yu cu ca h thng HACCP:

H thng HACCP bao gm 7 nguyn tc c bn nh sau: Nguyn tc 1. Phi phn tch c mi nguy hi: Xc nh cc nguy hI hoc tim nng nguy hI c lin qua tI tt c cc giai on sn xut thc phm; t khu sn sut nguyn liu x l, ch bin, phn phi cho n khu tiu th cui cng. nh gi kh nng d sy ra cc mi nguy hI v xc nh cc giI php kim sot chng. Nguyn tc 2.Xc nh cc Im kim sot ti hn( CCPs ): xc nh im/ th tc / cc bc thao tc ti cn c kim sot loi b cc mi nguy hi hoc hn ch mt cch kh d kh nng xy ra ca chng. Thut ng im y c th l mt cng on hoc mt giai on trong qu trnh ch bin thc phm, k t khu chun b nguyn liu th, tip nhn hay thu hoch, vn chuyn, thu gom, bo qun v.v. Nguyn tc 3: Xc lp cc ngng ti hn: Xc lp cc ngng ti hn m bo cc im kim sot ti hn (CCP) vn trong tnh trng c kim sot. Nguyn tc 4. Thit lp h thng gim st tnh trng c kim sot ca cc ngng ti hn: Xc lp h thng th nghim hoc quan trc nh k gim st tnh trng c kim sot ca cc im kim sot ti hn (CCP). Nguyn tc 5: Nu cc hot ng khc phc cn phi tin hnh khi vic gim st cho thy mt im CCP c th khng tnh trng c kim sot. Nguyn tc 6: Nu cc th tc thm tra khng nh rng h thng HACCP ang tin trin tt. Nguyn tc 7: Thit lp cc ti liu lin quan n mi th tc, mi bo co sao cho ph hp vi 6 nguyn tc trn v ph hp cho vic p dng chng. IV. p dng GMP v HACCP p dng HACCP thnh cng cn phi c s cam kt v tham gia ca lnh o v ton th mi ngi. N cng i hi s tham gia ca nhiu ngnh khoa hc nh ngnh V sinh an ton trong nng nghip, V sinh cy trng, ho hc, k thut xy dng dn dng.v.v. Vic p dng HACCP hon ton tng hp

vi vic p dng cc h thng qun l cht lng khc nh ISO 9000 v ISO 14000 - v bn thn n l mt giI php la chn mang tnh u tin v qun l an ton thc phm. Cc chuyn gia v cht lng cho rng cc c s ch bin thc phm nn p dng c GMP, HACCP v ISO 9000 nu c th, ba h thng ny s to ra ngI nh cht lng bn vng cho c s. C th v GMP l nn tng, ISO 9000 l nhng tr ct v HACCP l mi nh. Mt c c ch bin thc phm c iu kin p dng c ba h thng ny chc chn sn phm ca h s thng c trong cuc cnh tranh trn thng trng. Vic p dng HACCP c tin hnh theo cc bc sau y: Thit lp mt nhm hot ng v HACCP. M t sn phm. Nu r mc ch s dng. Xy dng lc tin trnh. Thm nh lc tin trnh ngay tI hin trng thc t ca qu trnh sn sut. Lit k tt c cc mi nguy hI c lin quan tI mi bc , tin hnh phn tch chng v cn nhc mi bin php kim sot cc mi nguy hi c ch ra. Xc nh cc im kim sot ti hn. Thit lp cc ngng ti hn cho tng im kim sot CCP. Thit lp h thng theo di gim st cho tng im kim sot. Thit lp cc hnh ng khc phc. Thit lp cc th tc thm tra. Thit lp phng thc ti liu ho v lu gi chng. Tt c cc doanh nghip v cc ngnh mun p dng thnh cng HACCP u phi tun th nhng nguyn tc v v sinh thc phm GMP. Nu iu kin c s h tng cng nh cc yu cu cp thit v khu v sinh cha c m bo th vic u tin l phi gii quyt cc khu trc. Chnh v iu ny m ngi ta gi GMP l h thng tin , cn HACCP l h thng b tr cho cc c s p dng GMP nhng mun lm tt hn. Mun p dng HACCP th cc c s bt buc phi p dng GMP trc. Khng c GMP th

khng th c HACCP. Khi nh gi v ghi nhn mc an ton thc phm c ci thin nh p dng H thng HACCP hoc ghi nhn vai tr ch o ca n trong ngnh cng nghip thc phm, ngi ta nhn thy rng vic p dng HACCP lun lun i hi mt phong tro ca c cng ng v i hi Chnh ph cn c mt vai tr ch o trong qu trnh thc hin HACCP. Theo o lut gn y nht ca hi ngh bn trn Uruguay v thng mi v thu quan (GATT), v Hip nh p dng cc bin php v sinh an ton cho cy trng v ng vt (SPS) cng nh Hip nh v hng ro k thut trong thng mi, xu th p dng cc qui nh ca ca U ban Codex ang rt c quan tm. Chnh v vy cc tI liu ca U ban Codex k c " cc ch dn p dng h thng HACCP" tr thnh ngun ti liu tham kho cho cc yu cu quc t v an ton thc phm. Vic p dng HACCP c xem l mt phn khng th thiu c trong cc ti liu ch dn ca Codex. p dng cc phng thc qun l mi ca quc t trong lnh vc qun l cht lng v sinh an ton thc phm t c cc yu cu ca GMP v HACCP s gip cc doanh nghip Vit nam iu kin thm nhp vo th trng thng mi th gii, tham gia cc Hip nh Thng mi trong AFTA v T chc Thng mi Th gii WTO.Trong nhng nm gn y, tnh trng ng c thc phm v s bng pht cc mi nguy t thc phm khc nh dch bnh b in, bnh heo tai xanh, l mm long mng, dch H5N1, d lng thuc bo v thc vt vt qu ngng cho php, sa, gia v c cht c hi l minh chng cho s cn thit phi c cc tiu chun lin quan n thc phm nhm bo v sc khe cng ng v gim thiu cc tc ng tiu cc v mt kinh t v x hi. ISO 22000 l g? ISO 22000 l tiu chun v h thng qun l an ton thc phm quc t, c chp nhn v c gi tr trn phm vi ton cu. Mt doanh nghip trong chui cung cp thc phm p dng v t c chng ch ISO 22000 c nhn nhn l mt n v c h thng qun l tt an ton v sinh thc phm v m bo cung cp cc sn phm thc phm an ton, cht lng cho ngi tiu dng. Tiu chun ISO 22000 c xy dng bi s ng gp ca 187 quc gia thnh vin trn th gii. Tiu chun ISO 22000 c ban hnh vo ngy 01/09/2005 v nm 2007 ti Vit Nam, c chnh thc tha nhn l tiu chun quc gia (TCVN ISO 22000:2007). Tiu chun ISO 22000 hin nay l t nguyn p dng. Tuy nhin cho d khng c quy nh bt buc p dng, th xu hng la chn ISO 22000 i vi doanh nghip sn xut, kinh doanh thc phm vn tr thnh ph bin. Mc tiu l gip cc doanh nghip ch bin, sn xut thc phm kim sot cc mi nguy v vt l (mnh kim loi, mnh thy tinh, g,

sn, ct, tc), ha hc (c t, kim loi nng, thuc bo v thc vt, ha cht), sinh hc (vi khun, nm mc, virus, k sinh trng) t khu nui trng, nh bt cho ti khi thc phm c s dng bi ngi tiu dng, nhm m bo an ton v thc phm. Ni dung ca tiu chun ISO 22000 Tiu chun ny nhm cung cp mt h thng qun l an ton thc phm ton din bao gm cc yu cu: Qun l ti liu h s, cam kt ca lnh o, qun l ngun lc, hoch nh v to sn phm an ton, kim tra xc nhn, xc nh ngun gc, trao i thng tin, ci tin h thng. Doanh nghip c th s dng cc ngun lc bn trong (nhn vin ca t chc) hoc cc ngun lc bn ngoi (t vn) p ng cc yu cu ny. Li ch khi p dng tiu chun ISO 22000 Mt doanh nghip p dng tiu chun qun l an ton thc phm ISO 22000 s c nhn nhn l c h thng qun l an ton thc phm t tiu chun quc t, to c li th cnh tranh cao, c bit to iu kin d dng cho vic xut khu sang cc th tng kh tnh trn th gii. Bn cnh , vic p dng ISO 22000 cn mang li nhiu li ch khc nh: - Gim gi thnh sn phm do gim chi ph x l sn phm sai hng, chi ph v thi gian nh gi th nghim. - C th c xem xt min, gim kim tra khi c giy chng nhn ph hp tiu chun ISO 22000. - p ng cc quy nh ca php lut hin hnh. - C c nim tin ca khch hng, ngi tiu dng v cng ng. - Tho mn nhu cu ngy cng cao ca ngi tiu dng v cht lng v an ton ca sn phm. Doanh nghip no nn p dng ISO 22000? Tiu chun ISO 22000 c th p dng cho tt c cc loi hnh doanh nghip sn xut, kinh doanh trong chui cung cp thc phm khng phn bit quy m, bao gm: - Sn xut v ch bin thc n gia sc. - Thc phm chc nng: cho ngi gi, tr em, ngi b bnh. - Doanh nghip ch bin rau, c, qu, tht trng sa, thy hi sn. - Doanh nghip sn xut, ch bin ung: nc ngt, nc tinh khit, ru, bia, c ph, ch,.. - Doanh nghip sn xut, ch bin gia v. - Cc hng vn chuyn thc phm. - Doanh nghip sn xut, ch bin n sn, nh hng. - H thng siu th, bn bun, bn l. - Doanh nghip sn xut vt liu bao gi thc phm. - Trang tri trng trt v chn nui. Cc bc trin khai h thng qun l an ton thc phm theo ISO 22000: Bc 1: Tm hiu tiu chun v xc nh phm vi p dng: Lnh o cn thu hiu ngha ca ISO 22000 i vi s pht trin ca t chc, nh hng cc hot ng, xc nh mc tiu v cc iu kin p dng c th. Bc 2: Lp nhm qun l an ton thc phm. Nhm ny bao gm Trng nhm v i din ca cc b phn trong phm vi p dng ISO 22000. Trng nhm an ton thc phm thay mt lnh o c s sn xut ch o p dng h thng theo ISO 22000 v chu trch nhim v lnh vc ny.

Bc 3: nh gi thc trng ca c s sn xut thc phm so vi cc yu cu ca tiu chun: Cn r sot cc hot ng, xem xt yu cu v mc p ng hin ti ca c s sn xut thc phm. nh gi ny lm nn tng hoch nh nhng ngun lc cn thay i hay b sung. Bc 4: Hun luyn, o to cho tng cp qun tr cng nh nhn vin vi cc chng trnh v hnh thc thch hp . Bc 5: Thit lp h thng ti liu theo ISO 22000, h thng ti liu c xy dng v hon chnh p ng yu cu ca tiu chun v cc yu cu iu hnh ca c s sn xut thc phm bao gm: Chnh sch an ton thc phm, cc mc tiu v an ton thc phm, cc qui trnh - th tc theo yu cu ca tiu chun, cc h s theo yu cu ca tiu chun, cc ti liu cn thit t chc thit lp, trin khai v cp nht c hiu lc mt h thng qun l an ton thc phm. Bc 6: Trin khai h thng qun l an ton thc phm: ph bin mi nhn vin nhn thc ng v h thng ti liu theo ISO 22000. Xc nh r trch nhim, quyn hn lin quan n tng qui trnh c th v hng dn nhn vin thc hin theo cc ti liu c ph duyt. Bc 7: Kim tra xc nhn h thng qun l an ton thc phm v chun b cho nh gi chng nhn bao gm: c s sn xut thc phm tin hnh cc cuc nh gi ni b, thm nh cc kt qu kim tra xc nhn ring l, phn tch kt qu ca cc hot ng kim tra xc nhn xc nh s ph hp ca h thng qun l an ton thc phm v tin hnh cc hot ng khc phc, phng nga cn thit. - La chn t chc chng nhn: C s sn xut thc phm c quyn la chn t chc chng nhn nh gi v cp chng ch. - nh gi trc chng nhn nhm xc nh mc hon thin v s sn sng ca h thng qun l an ton thc phm ng thi chun b cho cuc nh gi chnh thc. Bc 8: nh gi chng nhn do t chc c lp, khch quan tin hnh nhm khng nh tnh ph hp ca h thng qun l an ton thc phm ti doanh nghip vi cc yu cu tiu chun ISO 22000 v cp giy chng nhn. Bc 9: Duy tr h thng qun l an ton thc phm sau khi chng nhn nhm p ng yu cu ca tiu chun v khng ngng ci tin hng n tha mn yu cu ca khch hng v cc bn quan tm.

Quy trnh kiem tra phat hien Enterobacterhttp://www.hpastandardmethods.org.uk/documents/bsopid/pdf/bsopid16.pdf

Bn cnh cc pp phn lp c in th ngy nay vi s pht trin nhanh ca khoa hc k thut, ngi ta cn ng dng cc k thut mi, cng ngh sinh hc phn t vo vic pht hin cc lai VSV trong c enterobacter.Phn loi vi sinh bng Sinh Hc Phn TVietsciences- Dng Vn Hp, Nguyn Ln Dng 27/02/2007 Nhng bi cng tc gi 1. Khi qut v cc phng php phn loi vi sinh vt truyn thng: Trc y cng tc phn loi vi sinh vt vn da cn bn trn cc c tnh hnh thi, sinh l v ha vi sinh vt: nhum, hnh dng t bo khun lc, kh nng di ng, nhu cu dinh dng, kh nng sinh acid trong mi trng cng nh sc t to thnh v.v..Cc c trng ny i khi cng bc l nhng hn ch do cc c tnh c dng cho nhm vi sinh vt ny (Enterobacteriaceae) nhng li khng c ngha i vi nhm khc (vi khun gram m - gram negative bacteria). Hn ch ca cc phng php phn loi truyn thng dn n nhiu trng hp phi xc nh li tn phn loi ca mt s vi sinh vt. T trc n nay, n v c bn ca nh tn vi sinh vt l loi, bao gm nhm cc c th c mc tng ng cao v cc c im hnh thi. Cc phng php da trn cc phn ng sinh ha: T nhng hn ch ca vic xc nh cc c tnh hnh thi dn n nhiu nghin cu tp trung vo cc phn ng sinh ha c trng cho cc vi sinh vt ring bit. S khc bit ca cc phn ng c ngha cho phn loi cc vi sinh vt.

- API20E KIT, nguyn tc: da vo 20 phn ng khc nhau. Ni chung hin nay c nhiu ni vn dng k thut ny nhng nhn chung kt qu cng cn nhiu sai s do nhiu nguyn nhn khc nhau: c th trong cc trng hp gene quyt nh phn ng sinh ha nm trong plasmid li b mt do nhiu nguyn nhn khc nhau nh tui t bo, lng ging cy hay thay i trong qu trnh nui cy dn n sai khc v lm sai kt qu. - Phn bit bng thc khun th: Cc vi khun c mn cm vi thc khun th khc nhau. C thc khun th xm nhim lm tan t bo ngay lp tc sau thc khun th nhn ln thnh cc ht trong t bo ch, trong khi vi mt s vi khun th thc khun th xm nhim nhng li khng lm tan t bo vi khun v chng cng tn ti vi t bo vt ch. Da vo s khc bit ny m ngi ta dng cc thc khun th khc nhau phn bit cc i tng vi khun nghin cu. Tuy nhin, phng php ny cng bc l mt s nhc im kh gii quyt l cc c tnh mn cm ca vi khun vi thc khun th li thay i do iu kin ngoi cnh hoc l vi khun li c mc mn cm khc nhau i vi cc thc khun th khc nhau. Mt khc na, thc khun th rt d thay i cc c tnh do cng lm thay i c ch xm nhim vo vi khun ch. - Phn bit theo Typ huyt thanh: y l phng php c dng kh lu nhng rt hiu qu v hin vn ang c s dng (v d nhm vi khun Bt). Nguyn tc l da vo nhm quyt nh khng nguyn trn t bo vi sinh vt (b mt t bo tin mao hoc protein v). u th ca phng php ny l cc khng huyt thanh c dng bit ha nhiu chi khc nhau, trong nhiu trng hp c trng cho loi. Ni chung y l phng php kh n nh nhng hn ch ch yu ca phng php ny ch: yu cu k thut sn xut khng huyt thanh v tiu chun ha phn ng khng huyt thanh khng ng nht ti cc phng th nghim v tnh n nh gia cc ln lp li. - Phn bit bng loi hot cht khng khun (Bacteriocin): Bacteriocin bn cht l peptid khng khun sinh ra bi vi khun chng li vi khun khc. Nh vy, loi vi khun to ra loi bacteriocin no th c kh nng khng li chnh bacteriocin . Bi

vy c nhiu loi vi khun c phn loi da vo kiu bacteriocin. Kt lun: C nhiu phng php truyn thng c s dng trong nhiu nghin cu phn loi nhng khng c phng php no t ra vn nng thch hp cho mi i tng vi sinh vt, tnh chnh xc ch c th t c khi kt hp nhiu phng php khc nhau. 2. Cch tip cn phn loi hc vi k thut sinh hc phn t: Ngy nay nhng tin b trong sinh hc phn t m ra kh nng ng dng hu hiu trong phn loi hc v nghin cu a dng vi sinh vt. Nu nh cc phng php truyn thng ch tp trung trn mt s i tng vi sinh vt th phng php sinh hc phn t c th p dng trn mi i tng vi sinh vt. Ni chung cc phng php sinh hc phn t tp trung vo cc k thut ch yu l: + Phn tch acid nucleic. + Phn tch protein. + Phn tch lipopolysaccharid. + Ha phn loi hc. Trong phn ny chng ti tp trung gii thiu mt s k thut phn loi lin quan n acid nucleic. Cc phn sau chng ti ln lt gii thiu cc phng php lin quan n polysaccharid, lipoprotein v ha phn loi. 2.1. Phn tch acid nucleic: K thut phn tch acid nucleic lin quan ch yu n phn tch kch thc, cu trc acid nucleic, mi tng quan v trnh t acid nucleic thng qua gii trnh t ADN v lai ADN. a. Phn tch ADN plasmid: Phng php phn tch y bao gm tch plasmid, so snh cc loi plasmid v kch thc sau tinh sch plasmid v x l bng enzym ct hn ch, sau in di trn gel agarose v so snh s a hnh ca cc

mnh ct phn bit cc chng vi sinh vt vi nhau.

Plasmid vng

Streptomyces v Borrelia

Plasmid l nhn t di truyn ngoi nhn v sao chp c lp vi nhim sc th. Cu trc plasmid l si i ADN khp kn theo vng. Tuy nhin, c nhiu trng hp ngoi l l si kp ADN mch thng (i vi vi khun Borrelia v Streptomyces). Plasmid c tm thy hu ht cc vi khun v mt s t cc vi sinh vt nhn thc bc thp nh nm men. + Tch plasmid: Thng thng lng plasmid c trong t bo chim < 5% tng s acid nucleic ca t bo. Nh vy, ta phi thc hin php tch plasmid ra khi ADN ca nhim sc th. Ni chung c nhiu phng php tch plasmid t vi khun nhng nguyn tc chung l ph t bo vi khun dng enzym, siu m hay trong dung dch kim c cht ty ra l SDS hoc TritonX-100, sau l vic loi ADN ca nhim sc th v protein. Thng thng dng phng php kt ta vi axetat. Lc ny phn ln cc thnh phn ADN nhim sc th v protein ca t bo b kt ta b loi bng li tm v plasmid nm trong dch trn ta c tch khi kt ta vi ethanol hay isopropanol. y l phng php c hiu qu tch plasmid, trn thc t hiu qu tch cc plasmid c kch thc nh cao hn nhiu so vi cc plasmid c kch thc ln (>100 Kb). Vi cc plasmid c kch thc ln cn cc thao tc nh nhng trnh t gy do cc nguyn nhn c hc. Vi cch tch plasmid nh trn th sn phm thu c c ln cc on ADN c kch thc khong 500 bp v nhim ARN. trnh nhim ARN th cn x l vi RNase (ribonuclease A). Tuy nhin, c th tch theo cc phng php nh: - Tch bng Caeseium chloride. - Tch bng KIT QIAGENE. - Tch bng k thut khc. + in di plasmid trn gel agarose: Sau khi thu c plasmid th phi tin hnh kim tra trc khi phn tch kt qu in di trn gel agarose bit ph plasmid v kch thc ca chng. Khi tin hnh in di th nng gel khong 0.7- 1% vi mt trong cc m sau: TAE (40 mM Tris acetat 1mM EDTA, pH 8.0), TBE (89 mM Tris borat 89 mM boric acid, 2 mM EDTA, pH 8.0) v TPE (80 mM Tris- phosphats 2 mM EDTA, pH 8.0). Sau khi in di, gel c nhum vi ethidium bromide v pht hin di tia UV (310 nm). Nng ethidium bromide khong 5 mg/l v thi gian nhum l 10 pht. Sau c ra mt ln nhanh bng nc loi ion trc khi c nhn di UV v chp nh lm ti liu. Mt s vn cn lu khi phn tch plasmid: trn gel thng

thng plasmid c thy 3 dng: dng siu xon, dng t gy v dng mch thng khi b ct bi endonuclease. Dng di chuyn tc cao hn l siu xon (Hnh 1.1).

Hnh 1.1. Di chuyn ca plasmid mch thng (L) v siu xon (OC) khi in di i khi vic tch plasmid cng phc tp c bit trong nhng trng hp plasmid c kch thc nh c ln cc mnh ADN ca nhim sc th. Trong mi trng hp u c nhim cc RNA v chng di ng nhanh nht tr cc trng hp plasmid nh vy khng c nhm ln gia plasmid siu xon v dng chnh plasmid t gy v plasmid khc. Mt ch khc l trnh nhm ln khi so snh kch thc gia cc plasmid siu xon v dng dui xon hay mch thng. Thc t l ch c th so snh cc plasmid siu son vi nhau v kch thc. Khi tin hnh phn bit gia cc chng vi khun th ng nhin l ch c th thc hin so snh gia cc chng cng c plasmid vi nhau. Cng nn ch l khng phi cc chng u ging nhau v c tnh min dch nu c chung kt qu phn tch plasmid nh nhau. Php phn tch s c hiu qu trong cc mu c nhiu loi plasmid, tuy nhin cng phi tnh n vic cc plasmid cng c th b mt trong qu trnh bo qun. + K thut du vn tay (finger printing) phn tch plasmid vi enzym ct hn ch:

Cho n nay, ngi ta tm thy hn 400 enzym ct hn ch. Mc ch ca k thut ny b sung cho k thut so snh ph kch thc plasmid trn. Tc l ngi ta c th phn bit c s khc nhau ca cc plasmid c cng kch thc. Nh vy, khi x l plasmid vi mt hay kt hp mt s enzym ct hn ch th kt qu s thu c l cc on ADN c ct c kch thc khc nhau. Kt qu ny c lp li cao, do d s dng khi so snh cc mu khc nhau. Hin nay, c nhiu enzym ct hn ch c sn xut v tiu th trn th trng, cc enzym ny c im nhn bit thay i t 4-6 cp nucleotid. Thng thng x l enzym trong 1 gi sau mu c pht hin trn gel agarose hoc polyacrylamid Tu thuc vo kch thc ca cc mnh ct m s dng agarose c nng khc nhau: Bng 1.1. Nng agarose v kch thc mu ADN tng ng. Nng agarose (%) 0.3 0.5 0.8 1.0 1.2 1.5 2.0 Kch thc ADN (kb) 1-7 0.7-45 0.4-20 0.3-10 0.2-8 0.2-6 0.1-5

Theo kinh nghim ca nhiu tc gi (Grimont-1991) ph cc bng ADN c ngha cho so snh khng nn di 10 bng v nn c c bng kch thc nh v kch thc ln. Tuy nhin, cc bng ln khng nn vt qu 10 do bng c kch thc ln nh vy kh di chuyn trn gel. Trong cc trng hp so snh th nn dng thang chun ADN so snh kch thc v php phn tch du vn tay cho cc ln phn tch khc nhau. Nh trnh by trn, nu nh phn tch cc chng vi sinh vt da vo kch thc plasmid c ngha i vi cc i tng c nhiu plasmid th phng php x l enzym ct hn ch li c ngha ln trong cc trng hp nghin cu dch t hc trn cc chng c cng ph plasmid hay plasmid c kch thc ging nhau. Ngi ta ng dng k thut ny trong nghin cu chng E.coli (0157: H7) gy bnh t thc phm (Hamburger). Cc plasmid t cc chng khc nhau th c ph khc nhau v cc c im ct bi enzym ct hn ch. Kt qu to ra cc mnh ADN c trng cho c th lm c s cho php so snh. Tuy nhin kh c th so snh kt qu thu c t cc phng th nghim

khc nhau, s d nh vy l do khng dng cng mt loi enzym ct hn ch. Mt l do na cng cn c cp n l s xut hin cc t bin ngu nhin ti v tr enzym ct, kt qu ny to ra s khc bit v ph thu c t cc mnh ct. b. Phn tch ADN nhim sc th: Phng php phn tch y bao gm vic tch ADN ca nhim sc th v ct bng enzym ct hn ch phn bit gia cc vi sinh vt vi nhau. Mt ch khi tch ADN nhim sc th phi thao tc nh nhng trnh t gy do nguyn nhn c hc. Ni chung cc mnh ct nn c kch thc nh hn hoc bng 50 kb. Vic tch cc mnh ct c thc hin da vo k thut in di trong trng xung in (PFGE = Pulsed Field Gel Electrophoresis ).style="text-align: justify; margin-top: 6.0pt"> Nh vy k thut du vn tay khng ch p dng trong trng hp trn t bo mang plasmid m k thut ny cn c s dng vi nhim sc th cho mi trng hp.rng hp.ng hp.ng hp. Lin quan n vn ny phi k n k thut BRENDA: phn tch kt qu x l enzym ct hn ch vi vi sinh vt. y cch chn loi enzym ct hn ch v cng quan trng, theo cch chn ny c th to ra qu nhiu mnh ct nn khng th phn bit c cc bng ring r trong khi i vi cc trng hp khc li to ra qu t mnh ct c kch thc ln kh tch theo cc k thut in di hin c. Cc vi sinh vt khc nhau c t l GC khc nhau (dao ng t 25-75%) cc mnh ct thu c sau khi x l enzym ct hn ch rt khc nhau. Theo Nei M. v Li W. H. (1979) th s mnh ct c th thu c tnh trn l thuyt l: a = (g/2)r1 x {1-(g/2)}r2 Trong : g - t l GC ca ADN r1 - s cp GC r2 - s cp AT trong v tr ct ca enzym gii hn s dng a - s v tr ct khi s dng enzym ct hn ch. Mt khc, ngi ta cng cn c vo kt qu nghin cu cc v tr ct ca b gene vi sinh vt lm c s cho cch chn enzym ct. Thng thng cho mi php phn tch ngi ta ghi c s cc bng ct bi enzym v tnh ton kch thc cc mnh to thnh lm c s cho cc

php phn tch v sau. Tuy nhin, mt trong nhng nguyn nhn hn ch cho php phn tch trn l cc phng php tch ADN thng thng u dn n thay i kch thc do t gy ADN nhim sc th, mt khc s c mt ca plasmid ln cng to ra s khc bit gi trong kt qu phn tch, khc phc nhc im trn ngi ta phi thc hin php phn tch ADN nhim sc th nguyn vn pht hin cc ngun ADN tp nhim ln vo kt qu phn tch. + K thut in di trong trng xung in (in di trong trng in thay i, PFGE) - Nguyn tc: Trc tin cc mu a vo phn tch phi c x l trc bng loi enzym ct hn ch m c rt t im ct. Kt qu phi to ra c cc mnh c kch thc ln (50 kb 12 Mb), vi kch thc ny khng th in di theo phng php thng thng m ch c th tch ra bng k thut PFGE (t PFGE (t PFGE (ut PFGE (Pulsed-Field Gel Electrophoresis ). K thut PFGE ln u tin c Schwartz va Cantor (1984) gii thiu. Tuy nhin sau c nhiu tc gi m t li k thut ny, tuy c sai khc t nhiu nhng c s l thuyt ca cc phng php ny l nh nhau: Mc ch ca k thut ny l tng kh nng di ng ca cc mnh ADN c kch thc ln trong in trng, do cc thnh phn gel v m hu nh khng thay i theo cc phng php in di thng thng. Tuy nhin theo phng php thng thng th s in di lin quan n s di ng ca cc mnh ADN c kch thc khc nhau trn gel agarose trong mt trng in khng i. Kt qu y l phn t ln kh di ng hn phn t nh qua mt xch agarose, to ra s tch bit trong trng in di. Trong trng hp PFGE lc lm thay i kh nng di ng li l trng in tch thay i lin tc dn n cc mnh ADN c kch thc khc nhau thay i hng di ng. Nh vy kt qu l cc mnh c kch thc khc nhau s c kh nng di ng khc nhau. Kch thc cng ln th mc di ng cng chm. S di ng khc nhau ca ADN ch yu ph thuc vo kch thc ch khng phi do gel agarose nh phng php in di thng thng, hnh 1.2.

Hnh 1.2. S dng k thut PFGE phn tch nhim sc th sau khi x l vi Sma I vi 7 chng Haemophilus nluenzae. Tip theo cc m t ca Schwartz va Cantor, Smith v Codemine (1990) cp n s di chuyn ca cc mnh ADN khc nhau l hm s tuyn tnh vi kch thc ca chng. Trn c s c th iu chnh cc xung in v in trng. Tuy nhin cc nhn t khc cng c mi tng tc ln nhau v nh hng n kh nng di ng ca ADN nh: nhit , in th, nng agarose cng nh nng ion trong m. - Chun b ADN cho PFGE: Chun b ADN t nhim sc th khng ging nh cc phng php chun b ADN plasmid. Mt vn thng xy ra l s t gy ngu nhin ADN dn n sai khc trong kt qu phn tch. hn ch iu ny ngi ta phi thc hin k thut tch ADN NST khi t bo mu agarose c nhit nng chy thp LMT (Schwartz va Cantor 1984 v Smith, Klco 1988). Theo phng php ny hn dch t bo (dch nui cy hoc dch huyn ph) c trn vi LMT agarose ti 37oC. Hn dch u tin c x l vi enzym v cht ty ra tch ADN NST khi thnh, mng t bo, RNA v protein. Sau l x l vi EDTA, bt hot nuclease t bo vi proteinase K v Nlauroylsarcosine loi cc thnh phn khc ca t bo. Kt qu l phn LMT agarose c cha ADN NST c dng lm mu chy gel 0.51.0% (nn lm tan 65oC) v a ln mu chy gel PFGE, ti liu ca Smith (1988) m t chi tit cch chun b mu t cc t bo c v khng c thnh t bo: vi khun, nm men, nm si, t bo thc vt v ng vt. Ni chung vi cc trng hp c thnh t bo th cn n enzym ph thnh t bo. Lng ADN NST thng dao ng trong

khong 0.5-20 g ADN l thch hp. Nu qu nhiu ADN th khng th phn tch c, cn qu t th cng khng th pht hin c cc bng ADN trong kt qu phn tch. Mu ADN dng cho k thut PFGE c th c gi 1 nm trong lnh c cha 0.5M EDTA. - Sau khi thu c ADN NST trong mu LMT agarose th tin hnh x l vi enzym gii hn loi c t im ct. Tuy nhin mu u tin phi c x l vi PMSF bt hot protein K sau PMSF li phi loi b sau mt s ln ra vi cht ty ra v EDTA. Sau ch cn phn nh mu trong agarose c x l vi m v enzym ct hn ch qua m trong tube v sau ton b mu ny c s dng tch trong trng xung in. Bng 1.2 di y lit k cc enzym ct hn ch c dng cho phn tch PFGE. Bng 1.2. Mt s enzym ct hn ch c dng cho k thut PFGE. Enzym AatII ApaI ClaI MluI NarI NheI NotI NruI PvuI SacII SalI SfiI SmaI XhoI - ng dng ca k thut phn tch PFGE: K thut PFGE c s dng cho nghin cu trn nhiu i tng khc nhau (xem bng 1.3). Bng 1.3. Minh ho cc chng vi sinh vt c phn tch da trn kt qu PFGE thu c t cc on nhim sc th. V tr ct GACGT/C GGGCC/C AT/CGAT A/CGCGT GG/CGCC G/CTAGC GC/GGCCGC TCG/CGA CGAT/CG CCGC/GG C/TCGAC GGCC(N)4/NGGCC CCC/GGG C/TCGAG

Vi sinh vt nghin cu 1. Acinetobacter baumannii 2. Brucella spp 3. Campylobacter hyointestinalis 4. Campylobacter jejuni 5. CADNida arabicans 6. CADNida prapsilosis 7. Coxiella burnettii 8. Enterobacter cloacae 9. Enterococcus faecium 10. Escherichia coli 11. Listeria monocytogenees 12. Leptospira spp 13. Mycobacterium tuberculosis 14. Neisseria meningitidis 15. Psedomonas aeruginosa 16. Shigella spp 17. Staphylococcus aureus 18. Streptococci ssp

Ti liu tham kho Gouby et al (1992) Allardet, Servent et al (19980 Salama et al (1992) Yan et al (1991) Vasquez et al (1991) Carruba et al (1991) Heizen et al (1990) Haertl ADN BADNlow (1993) MirADNa et al (1991) Bohm ADN Karch (1992) Brosch et al (1991) Herrmann et al (1992) Zhang et al (1992) Bygraves ADN Maiden (1992) Boukadida et al (1987) Sodati ADN Piffaretti (1991) Prevost et al (1992) Single ADN Martin (1992)

Mi mt i tng c c trng ring v kt qu phn tch PFGE v c dng cho nghin cu so snh vi nhau v s tng ng. i vi nhiu i tng c nhim sc th th khng nht thit phi thc hin i vi mi nhim sc th m ch cn trn mt s nhim sc th m thi. V d trong trng hp ca C.albicans Monod (1990) ch cn thc hin php phn tch vi mt trong 4 t hp ca mt s nhim sc th l . Cho d theo cc cch chun b nhim sc th khc nhau th kt qu ca php phn tch PFGE u ging nhau. Php phn tch PFGE c

ng dng cho cc nghin cu mc loi vi loi. Mt s im cn lu khi s dng k thut PFGE: + K thut ny i hi phi c trang thit b chuyn dng, k thut cng nh kinh nghim v vic tch nhim sc th i khi gp kh khn trn mt s i tng vi sinh vt. + K thut PFGE khng nhy pht hin nhng sai khc nh gia cc mu, c bit khi thc hin vi mt enzym th kt qu c th ging nhau nhng khng chc chn l hai mu ging nhau hon ton. iu c ngha l k thut ny nn dng xc nh s khc nhau cn vic xc nh s ging nhau th khng tin cy. Tuy nhin ngay c khi xc nh s khc nhau i khi cc mu c plasmid nh hay thc khun th cng cn xt n v chnh ngun ADN ny cng to ra nhng sai khc gi. + S khc bit gia cc mu c th pht hin c khi dng cc enzym ct hn ch khc nhau. Tm tt: Phng php phn tch plasmid l phng php c dng ph bin trong cc phng th nghim chun on vi sinh vt. Vic kt hp gia phn tch plasmid vi s dng enzym ct hn ch s to c s phn tch chnh xc vi cc thng tin ng tin cy i vi cc mu phn tch. Tuy nhin, khi phn tch cc mu da trn plasmid th cn lu l cc plasmid c th b mt qua cc th h phn chia, dn n sai lch cc kt qu nghin cu. Khi thc hin php phn tch PFGE vi cc plasmid ln s khc phc c hn ch ca cc php phn tch hin ti vi plasmid nh nh trn. Phng php PFGE hin ang c dng rng ri ti cc phng th nghim vi sinh vt, p dng trn cc i tng d nui cy c th cho cc kt qu tt hn nhng hn ch v gi thnh thit b cng nh yu cu cao v k thut v kinh nghim. Mt khc khi s dng cc enzym ct hn ch him c th s kh pht hin c mc sai khc. Nh vy s kt hp gia cc php phn tch plasmid, PFGE v lai ADN s cho kt qu tin cy hn. 2.2. Lai ADN Nguyn tc c tin hnh nh sau: ADN tng s c tch ra v x l vi enzym ct hn ch (c trng hp khng cn x l vi enzym ct hn ch) sau in di trn gel agarose v chuyn ln mng lai.

Mu d (probe) c chun b v lai vi mng ADN trn. Kt qu php lai cho bit s khc bit gia cc mu ADN khc nhau. Php lai c tin hnh y da vo cu trc si i ADN t hai si n vi cc cu lin kt hydrogene theo nguyn tc b sung. Bt u l t gy cc lin kt hydrogene do ho cht (kim) hay bin tnh nhit, lc ny hai si n ADN tch nhau c gi l trng thi bin tnh ADN (denature). Nu ti thi im ny ngi ta cho vo mt ADN nh du bin tnh (mu d) sau to iu kin hi bin xut hin (renature), lc s cho php cc si n ca mu d bt cp vi cc si n ca mu ADN ban u (target ADN). Mc lai s ph thuc vo tnh c hiu (s tng ng) gia mu d v mu gc cng nh iu kin cho php lai thc hin (nhit , nng mui, pH, t ln mu d, kch thc mu d). Nh vy, vic chn iu kin chnh xc cho php lai c ngha rt quan trng cho tnh c hiu ca kt qu php lai. Sau khi lai, bc tip theo l ra bng m thch hp loi mu d d v cui cng l php o bc x nh du (tu theo phng php nh du phng x hay ho cht pht x hunh quang) nh gi mc tng ng ca php lai. Ni tm li mt s nhn t tham gia vo kt qu ca php lai l: mu d ADN, phng php nh du ADN, mu ADN lai v iu kin tin hnh v phng php nh gi kt qu php lai. + Chn mu d: Vic chn mu d c ngha quan trng cho php lai. Nhn chung cc nh phn loi hc vi sinh vt thng khng trc tip thit k mu d. V mt ny chng ti a ra mt s ni dung ch yu v tiu ch chn mu d theo Stahl v Amann (1991) nh sau: Mu d s lai vi ADN ch (target) v khng lai vi cc ngun ADN khc c mt trong mu lai. Khi phn tch cc mu vi sinh vt vi nhau th mu d nn l on nucleotid c trng cho cc mu phn tch phn bit vi cc sinh vt khc. Tuy vic chn mu d cng mang tnh kinh nghim, mu d i khi khng cn phi l ton b gene m ch l mt on gene m ho cho mt c tnh c trng cho i tng nghin cu (c th l yu t khng nguyn b mt, c t hay mt gene chc nng no trn plasmid). Vi cch chn mu d c kch thc nh (14-40 bp), c thun li l cc mu d c tng hp nhn to v php lai thc hin nhanh (di 30 pht) trong khi vi cc mu d ln thi gian ko di hn (khong 16 gi). Hn ch ln nht i vi cc mu d nh l kh khn khi nh du v dn n gim tnh c hiu ca php lai.

Mt cch thng c s dng l thit k cc mu d t cc chui ARN thng tin 16S. Cch chn c th cn c vo on ADN c mt trong cc i din mc di loi, trong loi hay thuc chi nghin cu. + Cc phng php nh du: Cc k thut nh du ADN c xem l lnh vc pht trin mnh nht trong sinh hc phn t. Ni chung k thut nh du c chia thnh k thut nh du trc tip v gin tip. Trong phng php trc tip th phn nh du pht hin c gn vo acid nucleic. i vi phng php gin tip th c mt phn chc nng c gn vo acid nucleic v phn ny li c pht hin gin tip da vo protein bm c hiu c pht hin bng k thut min dch. Sau y l chi tit cc phng php nh du. nh du trc tip: - Bng di y a ra cc loi c cht dng cho phng php nh du trc tip da vo vic nhn ADN c nh du ln s tng nhy so vi phng php nh du ch c thc hin vi cc mi n l. i vi cc phng php nh du trc tip th cc nhm to tn hiu c gn trc tip bng lin kt ho tr vi nucleic ca mu d. - nh du trc tip bao gm 2 bc chnh: to tn hiu da vo lin kt ho tr ca nhm tn hiu vi mu d v thc hin php lai gia mu nghin cu v mu d. Bng 1.4. Mt s c cht dng cho nh du trc tip. Ngun to tn hiu 32 P35

Ti liu Southern (1975)Collins& Hunsaker (1985) Renz&Kurz (1984) Albarella & ADNerson(1986) Amman & ctv (1988) Urdea ctv(1988) Heller& Shneider(1983)

S

Alkaline phophatase Ethidium Fluorescein Horseradish peroxidase Microperoxidase

Nitrobenzofuran

Draper (1984)

Tetramethylorhodamine

Amman&ctv(1990)

Hnh 1.3. Minh ho phng php nh du trc tip (A) v gin tip (B) nh du gin tip:

nh du gin tip c thc hin theo 3 bc: thc hin php lai gia mu d v mu ADN ch, phn ng gia nhm chc nng v protein bm c hiu vi nhm chc tn hiu thng bo (reporter) v cui cng l to ra tn hiu t nhm chc tn hiu. Bng 1.5. Lit k mt s h thng nh du gin tip. Nhm chc nng bm proteinNhm tn hiu (reporter) Ti liu dnLeary et al (1982) Biotin-dUTP/steptavidin Digoxigenein-dUTP/antidigoxigenein Sulphone/antisulphone Dinitrophenyl/antinitrophenyl Poly(dA)/poly(dT) Kessler et al (1990) Syvanen et al (1986) Leller et al (1990) Morrisey & Collins (1989)

Alkaline phosphatase Alkaline phosphatase Europium chelate Piruvate kinase Horseradish peroxidase

Acetyllaminofluorene/antiacetylaminofluorene

Alkaline phosphatase

Tchen et al (1984) Mc d c nhiu h thng nh du v pht hin tn hiu nhng kinh nghim cho thy h thng biotin v digoxidenin cho nhy cao hn c. Hai h thng ny c th pht hin ti mc di picrogam acid nucleic. Trong trng hp vi biotin th i khi c mc nhiu nn cao do biotin c mt trong cc sinh phm, cn i vi digoxigenein th c th khng gp kh khn ny. + nh du phng x v ghi phng x: Phng php nh du phng x l phng php c dng ph bin trong cc phng th nghim lai ADN vi cc thnh phn nh du l 32P v 35S. Kt qu ca php lai gia mu d v ADN ch c pht hin trn X-phim hoc nhp nhy lng. u im ni bt ca phng php nh du phng x l nhy c th t ti di mc picrogam ADN ch. Hn ch ca phng php ny l khng t c s thch hp cn thit cho cc th nghim chn on lin quan ti xc nh mu vi sinh vt, v d thi gian bn hu mu d ngn, nguy him cho ngi s dng

v cn yu cu an ton nghim ngt cho phng th nghim v c nhn s dng cng nh vic qun l cht thi. Xut pht t thc t trn m cng ngy ngi ta cng quan tm n cc phng php nh du phi phng x. Mc tiu l t c mt h thng nh du c nhy tng ng vi phng php dng cht phng x. Bng di y lit k cc yu cu l tng cho phng php nh du mu d: 1, D gn vi acid nucleic theo phng php n gin v c lp li cao. 2, Bn trong iu kin lai v bo qun. 3, Khng b nh hng v lm nh hng n phn ng lai. 4, C th thc hin vi iu kin phn ng lai trong dch th (liquid phase) hay c cht mang (solid phase). 5, Phng php pht hin nhy v n gin. 6, D b loi b sau phn ng lai. 7, D phn bit gia mu lai v mu cha lai trong cng iu kin. 8, C th ng dng vi h thng t ng ho. Tuy nhin, nu theo cc yu cu l tng trn th cha c h thng nh du phi phng x no tho mn. Hin nay, c nhiu h thng nh du phi phng x t nhy cao v l l do cc phng php ny ang c ng dng rng ri c bit l phng php da trn cc enzym nh Alkaline phosphatase, Horseradisk peroxidase. Tuy nhin phng php s dng cc cht phng x hin ti vn ang c dng cho mt s phng th nghim c bit. Chin lc nh du vi cht phi phng x. C 3 phng php nh du phi phng x: 1, Dng c cht ho pht x v sinh pht x, trong trng hp ny th c c cht ho v sinh pht quang u to ra cc bc x nh sng v

sau l phng php pht hin bc x ny. * Vi ngun c cht ho pht quang c loi nh AMPPD (Bronstein, Edward & Voyta, 1989) c th c dng trc tip nh l c cht cho enzym Alkaline phosphatase trong php lai ADN t nhy cao trong thi gian ngn (Bronstein et al., 1990). * Phng php pht x s dng enzym Alkaline phosphatase trn c s gii phng D-luciferin t c cht, v d D-luciferin-O-phosphate (Miska v Geiger., 1987). Hai phng php ny tng i nhy v ang c s dng rng ri. 2, Phng php pht hin da vo thay i mu: Phng php o mu c th thc hin trn mi trng dch th hay cht mang v kh nhy so vi phng php pht quang. Ngi ta to ra cc c cht sinh mu khi c mt enzym (chng hn nh Alkaline phosphatase). Li th ca phng php ny l vic pht hin n gin do kt qu l s thay i mu d pht hin v ng dng trong cc phng th nghim vi sinh vt. Mt phng php c th lm tng nhy ca phn ng mu bng cch thm mt enzym kch hot phn ng mu vo h thng. Mt v d cho iu ny l vai tr loi nhm phosphat ca phn t NADP thnh NAD do Alkaline phosphatase trong h thng pht hin mu d nucleic. Trong h thng ny th NAD c vai tr kch hot chu trnh oxy ho m c s tham gia ca alcohol dehydrogenease v diaphorase. Trong mi mt chu trnh th NAD s b kh thnh NADPH + H+. Phn ng oxy ho ny li km theo phn ng oxy ho m NADPH + H+ li b oxy ho thnh NAD, mi phn ng xy ra gn lin vi vic kh -indonitrotetrazolium mu tm thnh formazan. Sn phm cui cng formazan c nh lng bng php so mu (Self. 1985). 3, C cht hunh quang v pht x hunh quang theo thi gian. S dng c cht pht x hunh quang l phng php kh nhy v c th pht hin c ti mt phn t cht pht x (fluorescein). Ni chung vi cc mu sinh phm thng c mc nhiu tn hiu nn cao. Thc t ny c th c ci thin vi c cht fluorophore bn b kch hot bi sng nh sng. S pht x sau c ghi li khi cc bc x nn b gim. i vi phng php dng Alkaline phosphatase th vic pht hin nhn t gn lanthanide theo thi gian i km vi hot tnh enzym ny gi l phng php pht quang lanthanide khuych i bi

enzym (EALL: Evangelista, Pollack & Templeton, 1991). Trong trng hp ny, c cht khng c kh hnh thnh phc h gn vi lathanide pht x. Di tc dng ca Alkaline phosphatase th c cht v lanthalide b chuyn thnh phc h hot ng. Phng php ny kh nhy nhng hn ch ln nht ca n l cn thit b kch hot v o bc x hunh quang. + Qu trnh lai: Ni chung qu trnh lai (Hybridization ) c tin hnh theo mt trong 3 cch sau nh danh hay xc nh vi sinh vt: Lai vi vi sinh vt (in situ), lai trong dch th v lai vi cht mang (solid support). a. K thut lai vi vi sinh vt (in situ), hnh 1.4.

Hnh 1.4. Minh ho k thut lai in situ vi k thut FISH (fluorescence in situ hybridization) pht hin vi khun Helicobacter pylori. K thut ny c thc hin nh v chui acid nucleic trong vi sinh vt sau khi c c nh trong cc tiu bn hin vi. Tuy nhin, do hu ht cc vi sinh vt c kh nng thm (hay cho php) cc on ngn oligonucleotid nh du i vo t bo sau khi c nh mu v vy khi s dng mu d nh du vi cht nhum hunh quang c bit c th nhn thy c trc tip vi sinh vt di knh hin vi hunh quang. iu ng ch l phn ng lai phi tin hnh trong thit b c hn kn nhm hn ch vic bay hi ca dch lai. Vic bay hi dch lai ny dn n vic bm khng c hiu ca cht nhum hunh quang vi t bo. Nhit lai ph thuc vo oligonucleotid mu d. Sau phn ng lai th mu phi c xem ngay hoc c bo qun trong ti trong thi gian

6 thng. Nu mu d c hiu cho RNA th nhy tng ln rt ln v c ti hng ngn bn copy ca RNA trong mi t bo (Stahl v Amman, 1991). Lai trong mi trng dch th: Phn ng lai trong mi trng dch th xy ra nhanh hn nhiu so vi mi trng c cht mang pha rn (soild). Do c phn t ADN ch v mu d u c th di ng t do trong mi trng do phn ng t kt qu cao nht. Ni chung vi php lai thc hin trong dch th th thi gian kt thc nhanh hn t 5-10 ln so vi trong iu kin l cht mang (Bryan, et al., 1986). Tuy nhin, cng c th b sung thm cht mang lm tng phn ng lai. Cn thm bc cui cng l tch phc hp mu d-si ADN ch. Hn ch y l mu trong dch cho nn c th to ln cc nn km c hiu, do vy cn cc gii php lm hn ch mc nhiu ca nn cho thch hp.

Hnh 1.5. Minh ho k thut lai trong mi trng dch th. Trn thc t hu ht cc KIT thng phm dng cho vi sinh vt u tin hnh trong mi trng dch th. Vi sinh vt trong mu u tin c phn gii trong iu kin thch hp ADN ch lai vi mu d. Sau l bc lai v tch phc h mu d v ADN ch da vo cc thng s ca php lai theo kiu kp dp c th. Cc php lai theo kiu kp dp (hnh 1.9) cn c hai on ADN c trnh t khc nhau; mt on bm gi ADN ch gn vo cht mang v mt on l mu d.

iu quan trng l hai on ADN ny phi c trnh t khc nhau cho d chng u gn vi hai vng khc nhau trn on ADN ch theo nguyn tc b sung. Mu d nh du c b sung vo trong dung dch c cc on ADN ch nghin cu. Nh vy theo hnh v phc hp ADN mu d nh du pht hin gn vi ADN ch v phc hp mu d- ADN ch gn vo cht mang c hnh thnh. Phc hp ny c tch ra khi dung dch v khi c mt c cht thch hp pht hin c mu d nh du. Khi dng h thng pht hin da vo cc ho cht hunh quang hay to mu c th dng thit b pht hin kt qu mt cch t ng. + K thut lai da vo mng: Tc gi u tin gii thiu k thut c nh ADN trn mng l Nygaard v Hall (1963, 1964). Sau chnh Southern (1975) l ngi m t vic tch ADN khi gel sau khi in di v chuyn chng ln mng nitrocellulose. Cc on ADN ch c pht hin bng k thut lai v y l bc tin quan trng ca sinh hc phn t. T nm 1977 n nay c nhiu ci tin v php lai cng nh bc chuyn ADN ln mng ca cc tc gi khc nhau nh: Meinkoth v Wahl (1984). Ngi ta cng s dng mt s mng nynol, mng nitrocellulose hot ho hay c bn cho cc php lai. i vi cng vic nh loi vi sinh vt vi k thut c nh trn mng cho php lai ADN th cn chm mu (l ADN sch, hay t bo vi sinh vt hoc sinh phm c vi sinh vt nghin cu) ln mng thch hp. Mu c ly gii v ADN b bin tnh, sau ADN c c nh trn mng khi a vo t xy ti 80oC trong 2 gi hoc a vo x l vi tia UV trong trng hp s dng mng lynon. Mu d nh du sau c a vo dung dch. Bc tin lai c thc hin hn ch cc mu bm vo nhau khng c hiu. Sau l bc lai, mng sau khi lai c ra loi cc mu d cn d. Bc ra tip theo nh gi mc bm c hiu ca php lai. Sau cc mu d nh du lai vi v tr ADN mu trn mng c pht hin bng cc h thng pht hin tng thch. Ton b qu trnh lai trn mng c Meinkoth v Wahl (1984) m t chi tit. Khi pht hin typ vi sinh vt, chng ta phi s dng k thut Southern. Trong phng php ny ADN ca nhim sc th c tinh sch v c x l vi enzym ct hn ch thch hp, sau mu li c in di trn gel agarose v to ra du vn (finger print) ca nhim sc th. Sau khi in di gel c t di mng nitrocellulose hay mng nylon nm gia mt s lp giy. Lp giy lc pha trn c t trn cng phn gel v mng kp gia giy lc nh trong hnh 1.6.

Hnh 1.6. M t phng php chuyn ADN ln mng cho k thut Southern Blot. Kt qu l m t pha di c thm mt cch t nhin ln trn. iu gip cho vic chuyn cc mnh ADN t gel ln mng v bm cht vo mng vi kch thc v phin bn ng nh trn gel sau khi in di (m t chi tit theo Sambrook 1989). Phng php truyn thng ny cng c ci tin khi dng mng nylon chuyn ADN trong iu kin bin tnh (Read v Mann 1985). Thi gian chuyn c th vi gi hoc qua m. Vic c nh ADN trn mng c thc hin sau bng cch x l nhit hay tia UV nh trnh by trn. Tuy nhin, nhiu phng php ci tin c thc hin nhanh nh chuyn bng in di (Bittner, Kupfere, Morris, 1980) hay s dng bm chn khng (Medveczky, 1987; Olszewsk, 1988). Trong nhiu trng hp dng in chuyn ADN t gel agarose ln mng th trc tin gel phi c x l bin tnh v c lm trung ho tr li. Gel c t gia hai bn in c cc bn giy thm (hnh 1.7).

Hnh 1.7. Minh ho bc chuyn ADN ln mng. Sau ngun in c ni v lc ADN s c chuyn ln mng. Thi gian chuyn s ph thuc vo kch thc on ADN nhng thng dao ng trong khong 1-3 gi. Phng php c th thc hin theo chiu thng ng hay chiu nm ngang. Hin nay c mt s thit b bn trn th trng c dng cho k thut ny. Vi thit b nm thng ng, th ton b c ngm trong m v c th tng cng dng in (ch v c th lm tng nhit ), phng php ny cn dng nhiu m. Ngc li theo phng php nm ngang hay phng php semi-dry, y gel v mng c kp gia cc bn giy lc tm t bng m thch hp. Trng hp ny cn rt t m v cng dng in l 1mA/cm2 l thch hp. Trong trng hp chuyn ADN ln mng bng p lc do chn khng, vic thit lp h thng c trnh by m gel c t trn mng v s dng lc ht chn khng a ADN ln mng. Dng lc ht chn khng t c hiu qu chuyn ADN ln mng cao hn phng php thm t nhin. Hiu qu ti a cho chuyn gel vi trng hp geneom sau x l enzym ct hn ch c th t c sau 1 gi. + Phn tch RELPs vi k thut lai ADN. Gii thiu phng php: Nh trnh by, s phc tp trong k thut RELP to ra nhng kh khn cho vic xc nh cc tc nhn gy bnh trong cc nghin cu v dch t hc. iu ny cn kh khn v phc tp hn trong khi so snh cc kt qu thu c trn cc bn gel khc nhau hoc ti cc phng th

nghim khc nhau. Nguyn nhn l do s lng cc bng ADN ln ti mc khng th xc nh kch thc ca chng hay cc bng thu c khng lp li cc kt qu nghin cu. Mc d vy, theo phng php ny cc ph ADN phc tp sau khi x l vi enzym ct hn ch s c lm bin tnh v chuyn ln mng nitroxenluloza hay nylon. Mng s c lai vi mu d thch hp, kt qu ph php lai s r v khng qu phc tp do n ch hin th cc mnh ADN bt cp vi mu d. Vi mt s lng khng ln cc mnh hin th thu c sau php lai s cho php xc nh c chnh xc hn v kch thc. iu ny lm c s cho so snh gia cc gel vi nhau cng nh kt qu thu c t cc phng th nghim khc nhau. Khi s dng phng php ny cn ch mt s mu d sau: 1, Mu d c th l on RNA ribosom t mt loi sinh vt no : v d rRNA ca E.coli c th c cc cng ty thng phm cung cp (Boeringer Mannheim), y l mu d kh thun li v c nh du phng x hay vi cc c cht hunh quang. u im chnh ca mu d ny ch phn RNA l on kh bo th do mu d c th dng lai vi cc sn phm x l enzym ct hn ch ca nhiu loi vi khun khc nhau. C mt s loi khi lai vi mu d ch cho mt kt qu ging nhau trong khi loi khc khi lai cc chng vi mu d th li thu c kt qu khc nhau. y l c s cho phng php ribotyping. 2, Mu d c th l on ADN ngu nhin c chc nng khng xc nh (Tompkins et al., 1986). Mu d ny thng c dng cho cc loi c cha chnh cc on ADN ny. S dng cc mu d ny i khi rt c ngha trong vic phn bit cc mu sau khi tin hnh phng php ribotyping (Saunders et al., 1990). 3. Mt cch khc na cng c dng l s dng mu d l mt on ADN tch dng t mt gene bit. V d dng on gene m ho cho c t ngoi bo (exotoxinA) s dng nh typ Pseudomonas aeruginosa (Ogle et al., 1987). Mu d dng gene m ho cho c t Cholera c dng xc nh cc typ cho cc chng thuc h phy khun sinh c t (Yam, Li Lung & Ng, 1989). Vic s dng bt k loi mu d no cng to ra ph c trng ca php lai c ngha cho so snh gia cc chng vi nhau. Hn ch chnh ca k thut ny ch kt qu thu nhn ch khu tr phn gene bt cp vi mu d m khng c trng cho c h gene.

Bng 1.6. Kt qu thu c khi thc hin k thut RFLP vi dng cc mu d khng phi l ribosom. i tng Aeromonas spp Borrelia burgorferi Candida albicans Chlamydia trachomatis Corynebacterium diphtheriae Cryptococcus noeformans Escherichia coli Hemophilus influenzae Histoplasma capsulatum Lactobacillus helveticus Mycobacterium tuberculosis Pseudomonas aeruginosa Salmonella spp Staphylococcus aureus - K thut ribotyping: Nh trnh by trn mi mu d u cho cc kt qu c sc thuyt phc tuy nhin k thut dng mu d l on RNA ca ribosom a ra mt cch tip cn mi trong nghin cu dch t phn t vi cc vi khun c s khc bit ln trong khi cc mu d khc ch gii hn vi mt loi hay ch c ngha cho cc chng trong cng mt loi. K thut ny ln u tin c Grimont m t nm 1986 v nhanh chng tr thnh phng php hu hiu hin nay cho nghin cu dch t hc vi sinh vt mc phn t. Tnh hp l cho vic s dng k thut ny l ch gene m ho cho RNA ribosom c bo th cao. Cng c th pht hin thy nhng thay i cht t trong qu trnh tin ho i vi cc chui ADN trong cc vi khun nghin cu. Gene RNA ribosom c t chc thnh cc operon m cc gene ring r m cho cc RNA kch thc 5S, 16S v 23S chng c cch nhau bng cc on ADN spacer khng m cho gene no. Nu dng mu d hn hp gia 16S v 23S th kt qu php lai s hin th cc mnh tng ng vi phn ca gene ny trong khi dng mu d l on ca cc gene c tch dng c th a n kt qu hin th c phn gene tng ng v chui spacer. Nh vy s khc nhau ca Tc gi nghin cu v ti liu dn Altwegg an Luthyhottenstein (1991) Wallich et al (1992) Schmid et al (1992) Scieux et al (1992) Groman et al (1993) Spitzer (1992) Bohm ADN Karch (1992) Forbes et al (1992) Leathet al (1992) Delostreyesgavilan et al (1992) Mazurek et al (1991) Tompkins(1991) Sodati ADN Piffaretti (1991) Goh et al (1992)

kt qu ph thuc vo loi mu d s dng. - Yu cu k thut: thu c kt qu ti u cn c cc yu cu k thut c th cho tng bc thc hin. u tin l phi to ra c ph du vn tay thu c sau khi x l mu vi enzym ct hn ch. Ph vn tay ti u khi cc mnh ct phi c tch r v phn b ng u v kch thc trn gel. S lng cc mnh ADN thu c ph thuc vo mu ADN v loi enzym ct hn ch c s dng. Thng thng phi chn mt s mu x l th trc vi tng loi enzym (hay ng thi x l vi mt s enzym). C th thy r l cc enzym c 5 nucleotid ti v tr ct s to ra sn phm nhiu mnh hn cc enzym c 6 nucleotid ti v tr nhn bit. Thc t cng cho thy khi dng enzym ct hn ch (mt hay ng thi vi loi) thu c ph cc on ct hp l cho vic phn tch ribotyping th cc kt qu ny cng rt khc nhau. Nh vy, iu quan trng nn tin hnh trc cc php phn tch ny, phi chn c mt s chng c trng v th vi mt s enzym ct hn ch chn gii php cho nghin cu dch t hc vi k thut ny. Trong mt s trng hp kt qu phn tch khi ch tin hnh vi 1 hay 2 enzym ct hn ch c th khng a ra c kt qu chnh xc. Nh vy, khi c c kt qu hp l sau khi x l cc mu ADN vi enzym ct hn ch th tin hnh cc bc chuyn cc on ADN trn gel ln mng theo cc phng php c m t trn. Phng php chuyn bng chn khng l hp l hn c v kt qu cho vic chuyn cc bng c kch thc gn nhau li c tch ra sc nt trn mng. Khi thc hin php lai nn ch nu nh dng ngun ARN ribosom ca mt chng no nh du lm mu d th cn phi thay i iu kin lai nh nhit php lai c thc hin tt vi cc on ADN t cc chng vi sinh vt khc nhau. C mt s phng php nh du mu d l ADN ribosom. Phng php nh du phng x (Grimont, 1986), y l phng php c u th l ch cn vi bc n gin cho php lai v ra mu nhng li mang nhiu hn ch nh: thi gian bn hu ngn, yu cu an ton cho phng th nghim ring bit, nguy him cho ngi dng v gp kh khn vi vic x l cht thi phng x. Mi y c mt s phng php nh du mu d phi phng x c s dng kh thnh cng. Pitcher (1987) m t phng

php tng hp mu d gn vi bilatin pht hin ARN ribosom ca Providencia stuartii. Cht kch hot quang ho c Koblavi v Grimont m t (1990). ARN ribosom cng c nh du bng acetylaminofluorence (AAF) do Grimont (1989) m t. Cng ty Eurogeneetik (B) cung cp phc hp AAF-rARN trn th trng. Gn y Gustafero v Persing (1992) a ra mt phng php mi m y phc hp horseradisk-peroxysasepolyethyleneimine vi rARN v kch hot bng quang ho. Cc mu d c nh du bng cc cht phi phng x ny c th cho kt qu nhanh tng ng vi trng hp dng cc cht phng x. Nh vy kt qu tin hnh php lai cc mu ca cc chng khc nhau trong cng gel chuyn ln c th so snh vi nhau khi dng k thut ny. Trong trng hp xc nh chnh xc kt qu cc mnh lai vi mu d c th tin hnh so snh cc kt qu thu c t cc phng th nghim khc nhau. + ng dng k thut ribotyping: Xc nh typ vi khun bng k thut ribotyping c mt s u im so vi mt s k thut nh typ khc ch: Thc t l cc gene ca ribosom kh bn do chng nm trn nhim sc th. Hu ht cc vi sinh vt u cha nhiu phin bn ca operon ribosom do khi lai vi mu d thch hp th kt qu l to ra mt s mnh lai c kch thc khc nhau. Hin nay, ngun rARN ca E.coli nh du l mu d kh ph bin c thng mi ho.

Hnh 1.8. Kt qu ribotyping vi Helicobacter pylori. Do tnh a dng v phc tp ca k thut nh typ theo ribosom do trn thc t khi nhn kt qu bng mt thng kh c th pht hin c s khc nhau hay ging nhau gia cc chng trong cng mt loi khi tin hnh nghin cu dch t hc trn din rng. Owen v cng s (1992) nghin cu kh nng tr gip ca computer so snh kt qu thu c khi nghin cu cc chng Helicobacter pylori. Tng t nh vy Bialkowska-Hobranska v cng s (1990) dng thit b laze o mt cc mnh ADN lai cng vi s tr gip ca computer phn tch cc kt qu thu c t cc chng cu khun nha bo gram m. Phng php ny c th a ra s liu chung lm c s cho xc nh s ging nhau gia cc loi khc nhau. Cc phn tch thu c t cc s liu ca cc cc th khc nhau c th ch ra c cc c th mi lm c s cho nh danh cng nh theo di. Mi nghin cu cho thy mt iu quan trng v c ngha nht l cch chn enzym ct hn ch v loi mu d dng cho php lai (Saunder v cng s 1991). Tm tt: Nhn chung phng php lai ADN chng t u th ca n nh l mt k thut quan trng cho nh typ v nghin cu dch t hc nhiu loi vi sinh vt khc nhau. V nguyn tc phng php ny cng c u im v nhc im ca n.

u im: - S dng cho nhiu i tng vi sinh vt. - C cc mu d vn nng c bn rng ri trn th trng. Kt qu lai c lp li cao v kh n gin cho vic phn tch kt qu. - C th s dng s tr gip ca computer lu gi v phn tch kt qu th nghim. Hn ch: - Phng php s dng kh phc tp v tn thi gian. - Thng tin kt qu ch phn nh cho on gene lai vi mu d s dng. Hin nay, c th s dng cc on ADN tng hp lm mu d v iu ny thc t lm ci thin nhiu mt ca k thut lai nh: khi c ADN tng hp th khng phi thc hin cc k thut tch v tinh sch cc mnh ADN tch dng c th ln ADN plasmid. Mt khc khi lai vi mu d tng hp th phn ng tin hnh nhanh vi c hiu cao hn. Cho d k thut ny c s dng tt cho nhiu i tng vi sinh vt khc nhau nhng nm 1992 Heimberger v cng s tin hnh phn tch ADN c x l vi enzym ct hn ch trong trng xung in (PFGE), kt qu ny rt c ngha cho php phn tch su hn v phn bit cc chng vi sinh vt lin quan n s bng pht dch i vi cc chng vi sinh vt khc. Ribotyping v PFGE c chung nguyn tc da vo s phn b ca cc v tr enzym ct hn ch trn ADN ca ribosom. Tuy nhin, s khc bit ch ribotyping phn nh s phn b ca v tr cc enzym ct hn ch nm trong cc gene m ho cho rRNA hay nm trong vng nhim sc th c bo th cao, cn i vi PFGE phn nh s phn b ca cc enzym ct hn ch phn b trn ton b gene nghin cu. Nghin cu ca Prevost (1992) cho thy l k thut PFGE c th hiu qu hn k thut ribotyping i vi php phn tch cc chng Staphylococus aureus khng methicillin. 2.3. Nhn gene v k thut gii trnh t ADN. K thut lai ADN c s dng rng ri cho cc nghin cu xc nh typ ca nhiu i tng vi sinh vt. Ngun ADN ch i khi ch c s lng t tr khi cc tin hnh nui cy vi sinh vt. Trong mt s trng hp vic nui cy khng th thc hin c vi nhiu loi vi sinh vt hay virt hoc trong cc trng hp khc cn thi gian di nui cy vi sinh vt c ADN cho cc k thut lai hay nh typ. Kh khn ny

c khc phc bng cch lm tng ngun ADN ch mt cch nhanh chng bng k thut nhn gene PCR. a. K thut nhn gene PCR (Polymerase Chain Reaction) Vic nhn ADN cho php lm tng s lng gp hng triu hoc nhiu hn na i vi on ADN hay ARN nghin cu. C nhiu phng php khc nhau phn tch ngun ADN khuych i theo cch ny. Phng php n gin nht l s dng in di xc nh kch thc sn phm ca phn ng PCR. C th xc nh nhy v tnh c hiu cao hn nu sn phm PCR c lai vi mu d c hiu c nh du. Phng php a li thng tin nhiu nht l xc nh c trnh t ADN v ARN ca sn phm PCR. Vic xc nh c sai khc ca chui ADN s cho php xc nh v nh typ chnh xc nht cc i tng vi sinh vt nghin cu. Kt qu xc nh trnh t ADN cn cho php xc nh mi lin h tin ho v dch t hc ca cc chng vi sinh vt. - i nt v phn ng chui PCR: Phn ng chui PCR nhm khuych i ADN ln u tin c Saki (1985) gii thiu v y c coi l mt trong nhng tin b khoa hc quan trng nht v sinh hc trong thp k 80. Phn ng PCR s dng enzym chu nhit to ra nhiu phin bn theo hm s m. C th v d, ch cn 1 si ADN sau vi gi c th nhn ln 1011 phin bn ADN tng ng vi 100 ng ADN. Sau khi thc hin phn ng PCR th cn tin hnh mt s k thut xc nh sn phm, n gin nht l in di v xc nh kch thc ca sn phm PCR. Trong cc nghin cu chn on th kt qu ny rt c ngha, n chng t s c mt ca ADN ch trong mu nghin cu. C th nhn c tnh c hiu v nhy cao hn khi s dng k thut RFLP vi sn phm PCR hoc lai vi mu d c hiu. Tuy nhin, thng tin y nht vn l kt qu xc nh trnh t ADN ca sn phm PCR. Ngay t khi c gii thiu th PCR c xem l phng php a li kt qu kh nhy trong vic xc nh v pht hin vi sinh vt. Tnh u vit ca n th hin ch c th ch cn mt ht virus hay mt t bo vi khun no cng cho phn ng xy ra v khuych i ti mc c th pht hin c trong thi gian ngn. K thut PCR cn c dng nhn ADN t khun RNA sau khi c chuyn thnh cDNA sau phn ng phin m ngc (RT). Phng php ny nhanh chng c ch v tr ln ph bin cho cc nghin cu pht hin vi khun hay virus nng thp trong cc mu mi trng v bnh phm. V d vic pht hin mu mu nhim HIV c ngha l pht hin phn nh

ADN ca virus trong h gene ngi c kch thc gp 100 000 ln. Mt khc khi dng k thut min dch vi khng th th phi cn ti 8 ngy mi c p ng min dch trong mu, trong khi vi k thut PCR th ton b quy trnh pht hin ch mt vi gi (i vi ngun ADN hay RNA). Li th ca k thut PCR c tn dng cho vic chun b ADN lm mu d vi s lng ln cho cc php lai ADN trnh by trn. Cc mu d c th c nh du bng phng x hay phi phng x khi tin hnh phn ng khuych i. Mu d nh du vi k thut PCR c u th hn cc phng php nh du truyn thng bng k thut tng hp cc mch t gy (nick translation) hay nh du vi mi ngu nhin (rADN random primer labeling). Cc u th ny c th k n, c th nh l cn lng nh si ADN khun, c th tin hnh nh du vi cc mu d c kch thc ngn, chun b mu d khng cn tinh sch ADN khun. Bng 1.7. di y lit k mt s v d ng dng k thut PCR cho pht hin nhiu i tng vi sinh vt khc nhau. Rt nhiu cc kt qu nghin cu nh vy c trnh by trong cc tp ch chuyn ngnh nh: nh nh: nh nh: nh nh: Journal of Clinical Microbiology, Applied Environmental Microbiology. Bng 1.7. Mt s v d v ng dng k thut PCR trong xc nh vi sinh vt.Vi sinh vt Ti liu

Acanthamoeba sp Bordetella pertussis Borrelia burgdorferi Brucella sp Candida albicans Carnobacterium spp Chlamydia trachomatis Clostridium difficile Coxiella burneti Cryptosporidium parvum Cytomegalovirus

Vokin et al (1992) Houard et al (1989) Marconi ADN Garon (1992) Herman an Derider (1992) Miyakawa et al (1992) Brook et al (1992) Hayes et al (1992) Gumerlock et al (1991) Stein an Raoult (1992) Laxer et al (1991) Gozlan et al (1991)

Dengue virus Epstein-barr virus Erwinia amylovora Escherichia coli Frankia spp Gaeumannomyces spp Helicobacter pylori Hepatitis C virus Human immunodeficiency virus (HIV) Influenza virus

Henchal et al (1991) Samoszuk (1991) Bereswill etal (1992) Jackson (1992) Simonet et al (1990) Henson (1992) Claton et al (1992) Okamoto et al (1992) Dawood et al (1992) Claas et al (1992) Merien et al (1992)

Leptospira spp Niederhauser et al (1992) Litsteria monocytogenees Robertson et al (1991) Luteovirus Hackel et al (1990) Mycobacterium leprae Altamirano et al (1992) Mycobacterium tuberculosis Maiden et al(1992) Neisseria meningitis Charlotte et al (1993) Papillomavirus McOmish et al (1993) Parvovirus Barker et al (1992) Plsmodium falciparum Olsson et al (1993) Pneumocystis carini Yang et al (1991) Polio virus Seal et al (1992a)

Pseudomonas solanacearum Gage et al (1992) Rickettsia spp Taniguchi et al (1992) Rotavirus Rahn et al (1992) Salmonella spp Murakami et al (1991) Staphylococcus spp Vanevan et al (1991) Toxoplasma gondii Grimprel et al (1991) Treponema pallidum Riley et al (1992) Trichomonas vaginalis Breniere et al (1992) Trypanosoma cruzi Hill et al (1991) Vibrio vulnificus Nakajima et al (1992) Yersinia Sau y l nhng nt chung nht ca k thut PCR: Nguyn tc c bn ca PCR: + Khi qut chung: PCR l phng php dng enzym khuych i chn lc mt on ADN theo lut s m. Phn ng nhn gene c thc hin vi enzym ADN chu nhit, hai mi tng hp v 4 loi deoxyribonucleic (dATP, dGTP, dCTP v dTTP). Mi mt chu k phn ng nhn gene gm 3 bc (xem hnh v minh ho): Bc 1 l bin tnh ADN, c tc dng tch hai si n t si khun xon kp. Bc 2 l bt cp hai mi vo hai si n ca khun. Bc 3 l ko di chui theo mi, chiu 5-3 vi qu trnh trng hp gn cc dNTP dc theo si khun to thnh phin bn mi theo nguyn tc b sung. Tnh c hiu ca phn ng c quy nh bi mi v s sao chp trc tip theo trnh t si khun gia hai mi. Nh vy, kt qu to ra hng triu phin bn si khun trong vi gi. Mi cho phn ng l cc on ADN c kch thc 20-30 nucleotid c thit k theo trnh t ca on gene cn khuych i da theo d liu c sn. Mi cp mi phi hon thnh chc nng ca mnh trong

ton b phn ng, tc l chng phi bm c hiu vo ng v tr ring cho tng mi. S d nh vy l do nu chng khng bm vo v tr c hiu th khng th khuych i c on gene ch. Vi cc gene c bo th cao nh rADN th khi cp mi c thit k th chng c th nhn c gene t nhiu i tng. Ngc li, trong nhiu trng hp dng mi khng c hiu th c th nhn c cc sn phm PCR khng c hiu. Tuy nhin, c hai kt qu trn u c dng cho nh typ vi sinh vt. Th nghim phn ng PCR ln u tin dng mnh Klenow ca enzym ADN polymerase I t E.coli. Tuy nhin, enzym ny b bin tnh ti 94oC v nh vy cn phi b sung enzym mi sau mi chu k phn ng. n nay qu trnh ny c ci thin do dng enzym Taq ADN polymerase (Taq = Thermus aquaticus ) chu nhit. Enzym ny c tch ra t vi khun sng sui nc nng. Tc xc tc cho phn ng ca enzym ny rt nhanh, c th t khong 8000 bp/pht ti 755oC. Hot tnh enzym gim mt na khi x l ti 92.5oC trong 230 pht hoc 40 pht ti 95oC. Nh vy khi dng enzym ny th khng cn b sung enzym mi sau mi chu k phn ng. + Cc thng s k thut: Bt k mt phn ng PCR no cng bt u bng vic tch ADN lm si khun. Ni chung, theo l thuyt ch cn mt si khun cng cho phn ng to ra sn phm nhng trong thc t cn khong 10-100 ng/phn ng. i khi chun b ADN theo phng php n gin khng cn tinh sch cng ph hp cho phn ng PCR. Tuy nhin, iu quan trng l trnh s c mt ca EDTA (acide thylne-diamine-ttraactique) hoc m phosphat v kt qu s lm gim Mg++ (do EDTA hoc to thnh mui Mg3(PO4)2 khi nhit cao). Phn ng PCR c th nhn c on gene di 10 Kb nhng trong thc t thng dng nhn cc on c kch thc ngn hn (