Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

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CHNG I
GII THIU
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

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1.1. t vn
Cc bnh l nhim trng l nguyn nhn gy t vong hng u trn th gii.
Khng ch c nhng bnh nhim trng mi pht sinh m nhng bnh nhim trng c
gy cht ngi bit t lu cng ti xut hin. Hn na t l vi khun gy bnh
khng khng sinh ngy cng tng cao l nguy c ln cho sc khe cng ng. Nhng
bng chng gn y cho thy cc tc nhn gy bnh mc d rt khc nhau u s dng
nhng phng thc chung pht ng qu trnh nhim trng v gy bnh. Nhng c
ch ny to nn c lc (virulence) ca vi khun. Tm hiu cc c ch m vi khun s
dng xm nhp v gy bnh c ngha quan trng trong cuc chin chng li cc tc
nhn b nh ny.
Listeria monocytogenes l tc nhn gy bnh listeriosis. Vi khun ny c xp
l tc nhn gy bnh ng th ba thuc nhm B sau Streptococcus v E. coli. ng thi
l ngun chnh ly nhim bnh cho ngi trong cc sn phm bo quan lnh, vi sinh vt
ny c kh nng tn ti tng trng trong sn phm sut qu trnh bo qun lnh. i
vi vi sinh vt ng c thc phm khc, chng s pht bnh khi con ngi hp thu
liu lng, sau thi gian bnh cc triu chng lm sn biu hin. Trong Listeria
monocytogenes hin din vi s lng nh trong thc phm, khi vo c th chng
khng b o thi m tch ly ch c hi. Mc d bnh do Listeria monocytogenes gy
ra l tng s thp, 2 - 5 trng hp trn mt triu ngi mt nm, nhng t l cht do
vi khun ny l rt cao, 25 - 30% trng hp t vong trong cc ca nhim bnh.
i tng b nhim bnh do Listeria monocytogenes gy ra thng gp tr em,
tr s sinh, ngi gi, thai ph v ngi c h min dch km. Listeria monocytogenes
gy ra bnh nhim trng mu, vim mng no hoc st vim d dy rut, ng thi cng
l nguyn nhn gy ra tr cht sau khi sinh, non v sy thai ph n.
Do , vi tnh phn b rng v kh nng gy ra nhng tc hi nghim i vi
ngi b nhim L. Monocytogenes v c s chp thun ca khoa Mi trng v Cng
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

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ngh sinh hc, ti tin hnh thc hin ti Nghin cu v Listeria monocytogenes
trong cc sn phm thy sn.
1.2. Mc ch
Cu trc v c ch gy bnh ca Listeria monocytogenes.
1.3. Ni dung nghin cu
Kho st v cu trc ca Listeria monocytogenes.
Kho st v s phn b ca Listeria monocytogenes.
Tnh hnh nhim Listeria monocytogenes trong sn phm thy sn trn th
gii v Vit Nam hin nay.
Mt s phng php xc nh Listeria monocytogenes.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

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CHNG II
TNG QUAN












Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

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2.1. Tng quan v mt s vi sinh vt nhim trong thc phm
2.1.1. Salmonella sp

Hnh 2.1: Salmonella vi khun chuyn ch bnh thng hn

2.1.1.1. Phn loi
Gii: Bacteria
Nghnh: Proteobacteria
Lp: Gamma Proteobacteria
B: Enterobacteriales
H: Enterobacteriaceae
Chi: Salmonella
Loi: Salmonella sp
2.1.1.2. c im
Salmonella l trc khun Gram m. Hu ht cc loi Salmonella u c lng
xung quanh thn (tr Salmonella gallinarum v Salmonella pullorum), vy c kh nng
di ng, khng sinh bo t. C kch thc t bo vo khong 0,5 3 m.
Salmonella l vi khun hiu kh hay hiu kh ty nghi, thch hp 37
0
C, pH ti
u 7,2 - 7,6. mc trn cc mi trng thng thng.


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c im sinh ha:
Salmonella ln men glucose c sinh hi (tr Salmonella typhi ln men glucose
khng sinh hi) khng ln men lactose, Indol m tnh, Methyl Red dng tnh, VP m
tnh, Citrat thay i, Urease m tnh, H
2
S dng tnh (tr Salmonella paratyphi A: H
2
S
m tnh)...
Ln men sinh hi cc ng glucose, manit, sorbitol, ln men khng u
sacharose, khng ln men ng lactose, salicin, raffinose (T Minh Chu v Trn
Bch Lin, 1999).
2.1.1.3. Cu trc khng nguyn
Khng nguyn O
Mi Salmonella c th c mt hoc nhiu yu t khng nguyn. Hin nay ngi
ta bit c 67 yu t khng nguyn O. Vic xc nh cc yu t khng nguyn O l ht
sc quan trng nh nhm v nh type.
Khng nguyn H
Ch c nhng Salmonella c lng. Khng nguyn H ca Salmonella c th tn
ti di 2 pha.
Pha c hiu (phase 1): l nhng yu t c tnh cht c hiu cho
tng loi vi khun Salmonella, gm 28 khng nguyn lng c k hiu bng ch ci a,
b, c, g,.
Pha khng c hiu (phase 2): c k hiu s 1, 2, 3,.
(T Minh Chu v Trn Th Bch Lin, 2001)
Khng nguyn Vi
L khng nguyn b mt bao bn ngoi vch t bo vi khun, di dng mt
mng mng khng nhn thy c knh hin vi thng. Khng nguyn Vi ch c 2
type huyt thanh Salmonella typhi v S. paratyphi C.
Ngi ta da vo s khc nhau v cu trc khng nguyn xp loi Salmonella.



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Bng 2.1: Phn bit cc khng nguyn O, H, Vi:
Khng nguyn Tnh chu nhit
Tc ng ca
alcohol 50%
Formol 50%
O n nhit 2h30 100
0
C Khng B ngng tr ngng kt
Vi Bin nhit 1506 100
0
C Nhy cm Khng
H Bin nhit 2h 100
0
C Nhy cm Khng


Hnh 2.2: Cc khng nguyn b mt ca Salmonella.

2.1.1.4. Yu t c lc
C hai loi c t l ni c t v ngoi c t
Ngoi c t ng rut (entero toxin) c hai loi l LT v ST.
c t LT khng bn vi nhit, LT hot ha enzyme adenylcuclase trong
t bo nim mc rut, lm gia tng c-AMP (cyclo adenosine 5-monophosphate), c-AMP
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

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s kch thch tit Cl
-
v HCO
3
-
ra khi t bo, ng thi c ch Na
+
vo bn trong t bo.
Hu qu tch nc trong ng rut dn n tiu chy.
c t ST bn vi nhit, c ch tc ng tng t nh LT. ST hot ha
enzyme guanosylcyclase lm tng c-GMP (cycle guanosine 5-monophosphate) trong
t bo dn ti hin tng tiu chy.
2.1.1.5. Kh nng gy bnh
Salmonella xm nhp vo c th qua ng ming v hu ht l do n phi thc
n b nhim nh thc phm, sa, nc ung. Sau khi xuyn qua hng ro acid d dy,
vi khun di ng v pha rut non v sinh sn , tip tc chui qua 1 mng nhy v
vo thnh rut. Cc t bo Paneth ca nim mc rut tit ra mt loi peptide c tnh
chng li s xm nhp ca tc nhn gy bnh.
Salmonella nhim vo c th t hai ngun: t phn ngi hoc ng vt, t
ngi bnh. Trong phi k n tc ng ca ng vt lng v, trng v phn ca
chng lm cho vic lan truyn Salmonella d dng hn. Ngoi ra chut, mo, rui
cng l tc nhn gin tip dn n vic Salmonella lan rng ri hn khi phn ca chng
nhim vo cc thc phm khng c bo qun k. trong qu trnh git m cng cn
phng s nhim Salmonella nu khng thc hin ng quy trnh an ton thc phm.
Bnh thng hn

Hnh 2.3: Vim d dy rut
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nc ta, bnh thng hn ch yu do S. typhi, sau n S. paratyphi A, cn
S. paratyphi B v S. paratyphi C th t gp. Bnh ly t ngi ny sang ngi khc, qua
thc n, nc ung b nhim khun. Sau khi khi bnh v mt lm sng, khong 5%
bnh nhn tr thnh ngi lnh mang vi khun ko di hng thng hoc hng nm.
h, cha Salmonella l ng mt v vi khun vn c tip tc o thi theo phn ra
ngoi cnh. Ngi lnh mang vi khun l ngun lan truyn bnh quan trng.
Sinh bnh hc: Trc khun thng hn vo c th qua ng tiu ha n rut
non th chui qua nim mc rut ri vo cc hch mc treo rut. chng nhn ln v
mt phn vi khun b dung gii, gii phng ra ni c t. Ni c t kch thch thn kinh
giao cm bng, gy thng tn mng Peyer, xut huyt tiu ha, c th gy thng
rut. Ngoi ra, ni c t theo mu ln kch thch trung tm thn kinh thc vt no
tht ba, gy ra trng thi st ko di, li b, v gy ra bin chng try tim mch... T cc
hch mc treo rut vi khun lan trn vo mu gy nn nhim khun huyt v lan i khp
c th, ri vi khun vo mt v t quay tr li rut. Vi khun theo phn ra ngoi
cnh.
Cc bnh khc
Cc bnh khng phi thng hn do Salmonella gy ra thng l mt nhim
trng gii hn ng tiu ha trong cc trng hp nhim trng nhim c thc n m
Salmonella typhimurium... l tc nhn hay gp nht, sau l Salmonella enteritidis...
Nhim trng nhim c do Salmonella c thi gian nung bnh t 10 n 48 gi. Bnh
biu hin c st, nn, tiu chy. Bnh khi sau 2 - 5 ngy, khng c bin chng.
Ngoi ra, Salmonella c th gy nn cc tn thng ngoi ng tiu ha nh
vim mng no, th nhim trng huyt n thun, nhim trng phi...
Kh nng gy bnh ca mt s loi:
Salmonella typhi : Ch gy bnh cho ngi. nc ta bnh thng hn
ch yu do S.typhi gy ra.
Salmonella paratyphi A : Ch gy bnh thng hn cho ngi v cng hay
gp nc ta sau S. typhi.
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Salmonella paratyphi B : Gy bnh thng hn ch yu cho ngi, i khi
c sc vt. Bnh thng gp cc nc chu u.
Salmonella paratyphi C : Gy bnh thng hn, vim d dy rut v
nhim khun huyt. Bnh thng gp cc nc ng Nam .
Salmonella typhimurium v Salmonella enteritidis : Gy bnh cho ngi
v gia sc, gp trn ton th gii. Chng l nguyn nhn gy nhim trng, nhim c
thc n do n phi thc n nhim Salmonella.
Salmonella cholerae suis : Loi ny hay gy nhim khun huyt.
2.1.2. Campylobacter sp

Hnh 2.4: Phy khun Campylobacter
2.1.2.1. Phn loi
Gii: Bacteria
Nghnh: Proteobacteria
Lp: Gamma Proteobacteria
B: Eubateriales
H: campylobacteriaceae
Chi: Campylobactes
Loi: Campylobactes sp


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2.1.2.2. c im
Campylobactes l nhng vi khun b, mnh, Gram m, hnh du phy nhn hai
u, c kch thc 0,2-0,8m x 0,5-5m, rt di ng nh c 1 lng 1 u, khng sinh
bo t. Nui cy Campylobacters trn mi trng nhn to thng kh khn v vi khun
i hi iu kin vi hiu kh (micro-aerophile). Mc c 37
0
C nhng mc tt hn
42
0
C, khng mc 25
0
C. Thng dng cc mi trng chn lc nh thch mu
Columbia, thch mu tryptose phn lp vi khun. Campylobacters mc chm sau 2
n 3 ngy.
Bng 2.2: c im sinh ha phn bit loi v di loi ca vi khun
Campylobacter
Loi di loi
Pht trin Tnh cht sinh ha
25
0
C
42
0
C
1%G 3.5%N Catalase Nitrate H H2S
C. fetus ss vene + - - - + - - -
C. fetus ss fetus + - + - + - - -
C. jejuni - + + - + - + -
C.coli - + + - + - - -
C. cryaerophila + - - - + + - -
C. faecalis - + + + + + - +
C. lari - + + - + + - -
C. hyointestinalis + + + - + + - +
C. spu ss spu + + - - - + - +
C. spu ss bu + + + + - + - +
C. upsaliensis - + + - - + - -

Ch thch: 1%G = 1% glycerin
3,5%N = 3,5% NaCl
H = Hippurate hydrolysis sinh H
2
S trn mi trng TSI (< 3 ngy)
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C. fetus ss vene = C. fetus subsp venerealis
C. fetus ss fetus = C. fetus subsp fetus
C. Spu ss Spu = C. Sputorum subsp Sputorum
C. Spu ss bu = Sputorum subsp bubulus
Vi khun dng tnh Oxydase v Catalase, phn hy nitrat, H
2
S dng tnh,
khng oxy ha hoc khng ln men cc loi ng.
2.1.2.3. Cu trc khng nguyn
Cc nghin cu v khng nguyn ca Campylobacter phn ln da vo cu trc
ca loi C. jejuni. n nay xc nh c 3 loi khng nguyn l:
Khng nguyn b mt LPS: c bn cht l lipopolysaccharide, gm t nht
50 serotype bn vi nhit.
Khng nguyn lng H: c bn cht l protein vi hn 36 serotype
Khng nguyn mng ngoi OMP (Outer membrane protein): l loi
protein b mt chuyn bit v duy nht sn xut vacin. (Phan Hng Din, 2005)
2.1.2.4. Yu t c lc
c t ca Campylobacter gm hai loi c t: ni c t (endotoxin) v ngoi
c t (exotoxin). Trong ngoi c t c cha c t ng rut (enterotoxin) v c t
thn kinh (cytotoxin). Vai tr tc ng ca cc loi c t ny vn cha c bit r.
Liu gy c ngi l 500 1000 vi khun. Kh nng ca vi khun
Campylobacter ngi c l do s lng ln c t (H Huy Khi, 2004).
2.1.2.5. Kh nng gy bnh
Campylobacters l mt tc nhn gy vim rut cp tnh thng gp ngi.
Chng xm nhp vo ngi do n ung cc thc n v nc b nhim khun, do tip xc
vi ng vt, ch yu l vi gia cm, gia sc.
Thi gian bnh 2 - 11 ngy. Th lm sng thng gp l bnh cnh ca mt
vim rut cp tnh vi du hiu au bng, tiu chy d di, phn nc c khi c mu v
m. C trng hp xy ra nhim khun huyt. Tiu chy do C. jejuni ko di 5 - 10
ngy. a s trng hp khi khng cn iu tr bng khng sinh.
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C ch gy bnh cha r. Campylobacters c c t rut, nhng c kin cho
rng vi khun ny c kh nng xm nhp ging nh Shigella. Campylobacters nhn ln
ch yu hi trng v hng trng.
Bnh vim rut do C. jejuni xy ra nhiu ni trn th gii, gy tiu chy cp
ngi, c bit tr em v khch du lch t cc nc pht trin n cc nc nhit
i.























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Bng 2.3: Kh nng gy bnh ca Campylobacter.
Loi Ngun nhim Bnh trn ngi Bnh trn ng vt
C. fetus subsp fetus Tru, b, cu
Nhim trng mu,
sy thai, vim
mng no v bnh
d day rut
Sy thai trn tru,
b, cu v ri lon
tiu ha
C. fetus subsp venerealis Tru, b Nhim trng mu
Sy thai, cht phi
v v sinh
C. mucosalic Heo Khng gy bnh
Vim rut v hoi
t trn heo
C. cryaerophila
Tru, b, heo,
nga, cu
Cha xc nh
Trong phn gia sc
khe, i khi gy
cht phi

C. jejuni subbp deylei

Gia cm, heo,
tru, b, cu,
ch, chim, cn
trng, mo v
trong mi
trng
Bnh d dy rut,
sy thai, vim
mng no,vim
khp, hi chng
cuillian-barre
Bnh d dy rut,
vim v, sy thai
vo cui giai on
ca mang thai hoc
con yu hoc
cht vi ngy sau
khi sinh, g thng
l vt mang trng
C. jejuni subsp jejuni
C. Helvetius Ch, mo Khng gy bnh Bnh d dy rut
C. laridis
Gia cm, heo,
tru, b, cu,
ch, chim, kh
Bnh d dy rut,
vim nhim trng
mu
Bnh d dy rut
C. upsaliensis Ch, mo, ngi
Apse, nhim trng
mu
Bnh d dy rut
(Verkens, 1996)
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2.1.3. Escherichia Coli

Hnh 2.5: Escherichia Coli

2.1.3.1. Phn loi
Gii: Bacteria
Nghnh: Proteobacteria
Lp: Gamma Proteobacteria
B: Enterobacteriales
H: Enterobacteriaceae
Chi: Escherichia
Loi: e. Coli
2.1.3.2. c im
Hnh thi v c im nui cy
E.coli l trc khun Gram m, di ng do c lng quanh thn, mt s chng
E.coli c v polysaccharide, khng sinh bo t.
E.coli l vi khun hiu kh hoc k kh khng bt buc, pht trin d dng trn
cc mi trng nui cy thng thng, mt s c th pht trin c mi trng tng
hp n gin. Thng sng trong rut gi ca ngi v ng vt, theo phn i ra ngoi.
y l nhng vi sinh vt c kh nng pht trin nhit t 7 50
0
C. Nhit
thch hp 37
0
C, pH ti u l 4,4.
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Pht trin mnh trong mi trng Mac Conkey, EMB. Trn mi trng EMB, E.
coli cho khun lc c mu nh kim, trn, b u, ng knh khong ln hn 0,5m.
Trong mi trng lng, E. coli lm i mu mi trng, sinh hi hoc lm c
mi trng.
c tnh sinh ha
E.coli ln men nhiu loi ng sinh axit v sinh hi nh: Glucose, lactose,
ramnose; kh nitrat thnh nitric, gy nhy nht h hng thc phm, indol dng tnh,
methyl dng tnh, VP m tnh, citrat m tnh, urease m tnh, H
2
S m tnh.
2.1.3.3. Cu trc khng nguyn
Gm c cc loi khng nguyn
Khng nguyn O (somatic antigen): l khng nguyn ca thnh t bo, cu
to bi lipopolysaccharide. Cc c tnh ca khng nguyn O
Chu nhit, khng b hy 100
0
C trong 2 gi.
Khng b hy vi cn 50%.
B hy bi focmol 5%.
Rt c (1/20mg gy cht chut sau 24 gi)
Khng nguyn O gp khng huyt thanh tng ng s gy ra phn ng ngng kt
O. Khng nguyn O gi vai tr quan trng i vi kh nng gy bnh ca dng vi khun
c tnh cht chuyn bit cho tng loi vt ch.
Khng nguyn H (Flagellar antigen): c cu to bi protein, khi khng
nguyn H gp khng th tng ng s xy ra hin tng ngng kt H. khng nguyn H
c cc c tnh sau
Khng chu nhit.
B hy bi cn 50%.
B hy bi protease.
Khng b hy bi focmol 5%.
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Khng nguyn K (Capsular antigen): loi ny ch c mt s t vi khun ng
rut, c cu to bi polysaccharide hoc protein. Nu khng nguyn K che ph hon
ton thn vi khun s ngn cng phn ng ngng kt O.
Khng nguyn tim mao F (Fimbrial antigen): c dng hnh roi, di
khong 4m, thng hay xon, ng knh 0,7 2,1m, gip vi khun bm vo t bo
nim mc rut nn rt quan trng trong kh nng gy bnh ca vi khun.
Hin nay c khong 700 tuype huyt thanh ca E. coli t s t hp ca cc nhm
khng nguyn O, H, K, F. Da vo ngi ta c th nh danh vi khun.
2.1.3.4. Yu t c lc
Ngi ta chia E.coli thnh nhiu nhm, mi nhm sinh ra cc loi c t khc
nhau, hin c 5 nhm chnh : STEC (Shiga toxin-producing E.Coli) hoc VTEC
(Verotoxigenic E.Coli) v EHEC (Enterohaemorrhagic E.Coli), EPEC
(Enteropathogenic E.Coli), ETEC (Enterotoxigenic E.Coli), EAEC (Enteroaggregative
E.Coli) v EIEC (Enteroinvasive E.Coli)
Nhm STEC
STEC sn xut c t Shiga-like toxin (Slt), cn gi l Shiga toxin (Stx) hay
Verotoxin (VT). H c t Stx gm hai nhm chnh khng phn ng cho vi nhau l
Stx1 v Stx2. Trong khi Stx1 c tnh bo tn cao th Stx2 rt thay i v trnh t, to ra
nhiu subtype nh Stx2c, Stx2hb, Stx2e, Stx2g. Mt dng STEC c th sn sinh Stx1,
Stx2 hoc c Stx1 v Stx2, v thm ch nhiu dng ca Stx2.
Yu t khc c lin quan n c lc ca STEC l vic to ra enterohaemolysin
(EHEC-Hly) v c th c c t rut chu nhit EAST1. EHEC-Hly c m ha bi
gen trn plasmid 60 MDa (pO157) m plasmid ny c tm thy gn nh tt c cc
dng O157:H7 v cng kh ph bin c nhng dng STEC non-O157 na (Nataro v
Kaper, 1998). Trn plasmid ny c s hin din ca mt operon gm 4 khung c m
(open reading frame ORF) l hlyCABD. Trong hlyA l gen cu trc khi u cho
haemolysin. c t rut chu nhit EAST1 (u tin c m t nhm EAEC l
EAEC heat-stable enterotoxin 1), cng c tm thy nhiu dng STEC. Tm quan
trng ca EAST1 i vi kh nng gy bnh ca STEC vn cha c bit, nhng c
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mt vi trng hp tiu chy khng c mu m thng thy nhng ngi nhim
STEC
Nhm EPEC
Cc yu t c lc chnh bao gm gen eae m ha protein itimin cn thit cho
vic to ra tn thng dng A/E, plasmid 50 70 Mda ( EAF ) : m ha BFP ( bundle
forming pilus ), PER ( plasmid encoded regulator ) v Ler ( LEE encorded
regulator). Cc protein tit : Tir, EspA, EspB, EspD, EspF, EspG v MAP
(mitochondria associated protein), EAST 1 c kh nng ph hy t bo biu m v
CDT ( Cytolethan distending toxin )
Nhm ETEC
Nhm ETEC c hai nhm quyt nh c lc chnh l c t rut (enterotoxin)
v yu t nh v (colonization factor CF).
c t rut enterotoxin
Nhm ETEC gm nhng E. Coli to ra t nht mt trong hai loi c t ng
rut l ST v LT. ETEC gy bnh bng cch vi khun bm vo b mt mng nhy rut
non v tit ra c t rut, lm gia tng tnh trng tit dch. Nhm ETEC gy tiu chy
thng qua s tit c t ng rut LT v ST. E.Coli nhm ny c th ch tit c t
LT, hoc ch tit ST, hoc c th tit c LT v ST.
c t khng chu nhit (Heat-labile toxin - LT): c t LT ca E.Coli l
oligopeptide c lin h gn gi v mt cu trc v chc nng vi c t t (cholera toxin
- CT) do Vibrio cholerae tit ra. LT v CT ging nhau nhiu c tnh nh cu trc, trnh
t acid amin (ging nhau khong 80%), tng ng v receptor, hot tnh enzym, v tc
ng ca n trn th hay nui cy t bo.
Sau khi c t i vo ni bo, chng di chuyn trong t bo nh h thng vn
chuyn ca Golgi. ch n ca LT trong t bo l enzym adenylate cyclase nm lp
mng ngoi ca t bo biu m rut. Peptide A1 c hot tnh ADP-ribosyltransferase
chuyn phn ADP-ribosyl t NAD n ca protein lin kt GTP (GTP-binding protein)
l GS, gy hot ha enzyme adenylate cyclase, lm gia tng AMP vng (cAMP) trong t
bo. V vy enzyme cAMP-dependent protein kinase (A kinase) c hat ha dn n
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 19

s phosphoryl ha knh chloride (Cl
-
) mng t bo biu m vt qu mc bnh
thng. Kt qu dy chuyn l kch thch t bo bn di tit Cl- v ngn cn s hp th
NaCl bi nhng t bo c lng nhung. Hm lng ion trong lng rut gia tng ko theo
s di chuyn th ng ca nc t t bo vo lng rut, gy tiu chy (Nataro v Kaper,
1998). Mc d s kch thch ca Cl
-
do s gia tng lng cAMP trong t bo l cch gii
thch c in v c ch gy tiu chy ca LT v CT, ngy cng c nhiu bng chng cho
thy rng p ng tng tit i vi nhng c t ny c c ch phc tp hn. Mt c
ch tc ng khc ca c t c lin quan n nhng prostaglandin E (PGE1 v PGE2)
v yu t hot ha tiu cu. S tng hp v phng thch nhng cht chuyn ha ca acid
arachidonic nh prostaglandin v leukotriene c th kch thch s vn chuyn cc cht
in gii v kch thch nhu ng rut. C ch tc ng khc th hai c lin quan n h
thn kinh rut (enteric nervous system ENS) iu ha nhu ng v s tit ion rut.
C ch th ba l CT v LT gy p ng vim rut dng nh.
c t chu nhit (Heat-stable toxin - ST): Ngc vi LT, ST c trng
lng phn t nh v nhng cu ni disulfur ca n gii thch cho kh nng chu nhit
ca c t ny. ST c chia thnh 2 nhm l STa v STb, khc nhau v cu trc v c
ch hot ng. Gen m ha cho c 2 nhm c tm thy ch yu trn plasmid v vi
gen m ha ST cng c tm thy trn transposon. Sta (hay cn gi l ST-I) c to
bi ETEC v mt vi vi khun Gram m khc nh Yersinia enterocolitica v V.
cholerae non O1. ST ging 50% trnh t acid amin vi c t chu nhit EAST1 ca
EAEC. Gn y, cn c bo co cho rng mt vi dng ca ETEC cng c th sn sinh
c t EAST1 ngoi c t STa. Cn STb ch c tm thy ETEC.
Yu t nh v (colonization factor CF): C ch m ETEC kt
dnh v c tr trn lp mng nhy rut c nghin cu k. gy tiu chy, ETEC
u tin phi kt dnh vo t bo rut non nh vo lng trn b mt ca vi khun, gi l
yu t nh v (CF). CFA c th c phn loi da trn c tnh hnh thi. C 3 loi
chnh gm loi lng hnh que cng, lng hnh que mm c dng b, lng c cu trc
mnh mm. Gen ca CFA thng c m ha trn plasmid, cng l ni m ha cho
c t ST v/hoc LT.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 20

Nhm EAEC
Nhng yu t c lc chnh ca EAEC bao gm cc dim bm dnh kt tp AAF
(aggregative adhesion fimbriae), yu t iu ha bm dnh kt tp aggR, protein Pet v
c t EAST 1 (enteroaggregative heat stabe toxin 1). AFF c xem l yu t
quyt nh c lc. Ba loi AAF c bit n gm AAF/I, AAF/II v AAF/III,
trong loi I v loi II c cu trc b, loi III c cu trc dng si ring bit. Cc AAF
to nn kiu bm dnh hnh chng gch ln t bo Hep 2. Yu t aggR c vai tr iu
ha s biu hin ca cc AAF. Protein Pet c tit ra mng ngoi vi khun gy ra s t
dch v gy c cho biu m tiu ha. EAST 1 c kh nng ph hy t bo biu m.
Ngoi cc yu t c lc nu trn, EAEC cn tit ra mt protein lm tan mu v lm
mt thng bng vn chuyn protein qua mng. Cc yu t c lc nu trn ca EAEC
ch yu c m ha bi cc gen nm trn plasmid c phn t lng 60 MDa. Mt s
yu t c lc c m ha trn cc gen trn nhim sc th ang c nghin cu
Nhm EYIC
EIEC gy bnh ch yu do kh nng xm nhp vo nim mc i trng. Kh
nng xm nhp c m ha bi gen trn plasmid 140 MDa. Cc gen trn plasmid ny
m ha cho cc khng nguyn xm nhp (IpaA n IpaD, Ipa : Invasion plasmid
antigen). EIEC cn c kh nng sn xut c rut ging mt s Shigella. Gen m ha
cho c t ny c tn l sen (Shigella enterotoxin), c ch gy bnh ging vi khun l.
EIEC xm nhp vo trong t bo biu m i trng, lm tiu cc ti thc bo v nhn
ln trong bo tng, ph hy t bo v xm ln sang cc t bo khc.
2.1.3.5. Kh nng gy bnh
Kh nng gy bnh rt a dng: gy nhim khun ng tiu, vi c th yu th
gy nhim khun mu, gy vim mng no tr s sinh, gy tiu chy.
Bnh tiu chy do E.coli
Cc E.coli gy bnh tiu chy ngi gm c:
Enterotoxigenic E.coli (ETEC)
L loi E.coli sinh c t rut. ETEC l mt nguyn nhn quan trng gy tiu
chy nng ging triu chng do Vibrio cholerae 01 gy ra ngi. Bnh tiu chy do
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 21

ETEC xy ra ch yu cc x nhit i v c th gp cc la tui khc nhau, nhng
c bit tr nh thng thy bnh cnh lm sng nng d dn ti tnh trng kit nc
v ri lon in gii.
ETEC cn l mt nguyn nhn thng gy tiu chy cho khch du lch t cc
nc pht trin sang cc nc ang pht trin.
C ch gy bnh: ETEC vo rut s gn vo nim mc rut nh cc yu t bm
dnh, ng thi sn sinh ra c t rut tc ng ln t bo nim mc rut gy xut tit ra
mt s lng ln mt cht dch ng trng vi huyt tng. Bnh nhn nn, tiu chy
lin tc, phn ln c au nhc bp c, au bng v st nh.
C hai loi c t rut c nghin cu k v tnh cht sinh l, sinh ha v
tnh truyn bng plasmid l :
c t rut LT (Heat - labile)
c t rut ST (Heat - stable)
Nhng chng ETEC c th sinh ra mt hoc hai loi c t rut ty thuc vo
plasmid m chng mang.
LT loi c t rut b hy bi nhit, l mt protein gm 2 tiu phn A (Active) v
B (Binding) c chc nng ring bit. Tiu phn A c hai tiu n v A1 v A2, tiu phn
B c 5 tiu n v B1, B2, B3, B4 v B5. Cc tiu n v B c chc nng gn vi th
th ganglioside GM1 b mt t bo biu m rut v chun b m ng cho tiu phn
A m ch yu l A1 xm nhp vo bn trong t bo. Tiu n v A tc ng ti v tr
ch mt trong mng bo tng ni iu h.a enzymee adenylate cyclase. Adenylate
cyclase b hot ha v lm tng hm lng adenosine monophosphate vng (AMP
vng). Hin tng ny dn ti s tng thm ca cc in gii v nc qua mng rut,
gy tiu chy cp v kit nc, ri lon in gii.
ST loi c t rut khng nhit, l mt phn t c trng lng thp nht v khng
c tnh khng nguyn. Th th dnh cho ST khc vi th th LT. Sau khi c gn vi
th th, ST s hot ha guanylate cyclase trong t bo nim mc rut. Hin tng ny
dn ti s tng guanosine monophosphate vng (GMP vng) v do xy ra tnh trng
tng tit dch rut.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 22

Enteropathogenic E.coli (EPEC)
EPEC hin nay c bit gm mt s type huyt thanh thng gy bnh tiu
chy cp (bnh vim d dy - rut) tr em la tui nh (tr di mt tui), c th gy
thnh dch. Cc v dch do EPEC thng hay gp trong bnh vin, c ch gy bnh ca
EPEC cha c bit r. Cc EPEC phn lp t cc v dch thng l thuc cc typee
huyt thanh:
O26 : B6 O111 : B4 O126 : B16
O55 : B5 O119 : B4 O127 : B18
O86 : B7 O125 : B15 O128 : B12
Enteroinvasive E.coli (EIEC)
L loi E.coli gy bnh bng c ch xm nhp t bo biu m nim mc rut, gy
tiu chy ngi ln v tr em vi nhng triu chng bnh l. ging Shigella: ngha l
au bng qun, mt rn, i tiu nhiu ln, phn c nhiu mi nhy v mu.
Ngi ta chng minh c rng kh nng xm nhp t chc rut ca EIEC
c chi phi bi plasmid. EIEC c th khng ln men lactose, khng di ng v ging
Shigella v nhiu mt k c cu trc khng nguyn. Do vy nhng v dch tiu chy do
EIEC gy nn d b ln ln vi tiu chy do Shigella.
Enteroadherent E.coli (EAEC)
L loi E.coli bm dnh ng rut gy bnh do bm vo nim mc v lm tn
thng chc nng rut.
Enterohemorrhagic E.coli (EHEC)
EHEC l mt trong nhng tc nhn gy tiu chy c th dn ti vim i trng
xut huyt v hi chng tan mu - ure huyt. EHEC l nhng chng E. coli c kh nng
sn xut mt c t gy c t bo Vero (Verocytotoxin), gi l VT.
Cc nhim khun khc do E.coli
E. coli c th gy nn nhim khun ng tit niu: s ng nc tiu do si,
thai nghn... to iu kin thun li cho bnh nhim khun ng tit niu d xy ra .
Mt khc, khi thng niu o, ngi ta c th gy ra nhim khun ngc dng.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 23

E. coli c th gy ra nhim khun ng sinh dc, nhim khun gan mt, vim
mng no tr cn b, nhim khun huyt ...
2.2. Gii thiu v Listeria monocytogenes
2.2.1. Lch s pht hin
L. monocytogenes ln u tin c m t bi E.G.D Murray vo nm 1926 da
trn su trng hp t t tr. Murray gii thiu Vi khun monocytogenes trc khi
JH Harvey Pirie thay i tn chi l Listeria nm 1940. Mc d L. monocyotogenes
nhim c ng vt v con ngi c ni n vo nhng nm 1920, nhng cho n
nm 1952 ti ng c cng nhn L. monocyotogenes l mt nguyn nhn quan
trng gy ra nhim trng v vim mng no ca tr s sinh. ngi ln mc bnh do vi
khun ny nguyn nhn l do tn thng h thng min dch, chng hn nh ngi lm
dng thuc c ch min dch v Corticosteroid cho khi u c tnh hoc cy ghp ni
tng, v nhng ngi nhim HIV.
Mi cho n nm 1981 L. monocytogenes c xc nh l mt nguyn nhn
gy bnh t thc phm, lin quan n 41 trng hp mc bnh v 18 ca t vong, ch
yu ph n mang thai v tr s sinh. T mt s trng hp nhim Listeria t thc
phm c bo co, v L. monocytogenes c cng nhn rng ri nh l mt
mi nguy him quan trng trong ngnh cng nghip thc phm.
Listeria c xem l vn nghim trng nhim vo cc sn phm tht v tht gia
cm vo nhng nm 1980. Nhng nm 1990, B Nng nghip M cho bit c mt trn
dch bnh ny t bnh m kp xc xch nng v c th l cc ca hng bn tht ngon dn
n t nht 101 ngi b bnh khin 15 ngi ln t vong v 6 thai nhi b sinh non.
Nm 1999, loi L. monocytogenes c bit cc c tin trin bo ng cc
vin chc y t v h buc nhng nh sn xut thc phm phi gii quyt vn . Mc d
quy nh tm thi i vi cc sn phm tht v tht gia cm ch bin sn pht hnh
nm 2003 gip kim sot s bng pht vi khun, nhng r rng khng th loi b
c bnh hon ton. Theo Trung tm y t d phng CDC, c tnh 2.500 ngi b
bnh listeriosis nng mi nm, trong s c 500 ngi t vong. Ngi c nguy c cao
c th mc bnh ny sau khi n thc phm thm ch ch nhim vi con vi khun.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 24

Nm 2006, trong mt n lc bo v cng ng, C quan lng thc v dc
phm FDA ph chun tn thnh vic s dng thuc xt kh trng gip gim nhim
khun cc sn phm tht v tht gia cm ch bin sn. Thuc phun bao gm 1 hn hp
gm 6 loi virus v hi dit khun L. monocytogenes.
2.2.2. Phn loi
Theo George M. Garrity, Julia A.Bell v Timothy G.Lilburn, phn loi hc ca
Listeria spp. Trong gii vi sinh vt nh sau:
Gii: Bacteria
Ngnh: Fitmicutes
Lp: Bacilli
B: Bacillales
H: Listeriaceae
Ging: Listeria
Lc u Listeria ch c bit n vi mt hay hai loi ch yu l Listeria
monocytogenes, v sau th xc nh c 6 loi gm:
Listeria monocytogenes
Listeria innocua
Listeria seeligeri
Listeria ivanovii
Listeria grayi
Listeria welshimeri
Cc loi c tnh tng ng v di truyn nn gip gii thch c kiu hnh tng
t nhau gi cc loi. Kh nng tan huyt l c tnh nhm phn bit gi cc loi.
n nay ch c loi L. monocytogenes cho thy l tc nhn chnh gy bnh cho
ngi v ng vt, khi L. invanovii chi gy bnh cho ng vt c bit l th.




Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 25

2.2.3. c im

Hnh 2.6: L. monocytogenes.
2.2.3.1. c im chung
L. monocytogenes l trc khun Gram dng, k kh ty nghi pht trin nhit
t 1 - 45
o
C, khng to bo t nhng c chuyn ng in hnh khi c cy nhit
20 - 25
o
C v c th pht trin trong t bo .
L vi khun hnh que mnh, chiu ngang khong 0,5m, chiu di khong 1-
2m. Phn ng catalase dng tnh. Sau khi nhum Gram ta thy nhng t bo hnh que
ring l hoc nhng t bo ni vi nhau thnh mt chui t bo.
Vi khun ny c th tn ti pH rng t 4,3 - 9,6.


Hnh 2.7: T bo vi khun L. monocytogenes Gram(+)
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 26



Hnh 2.8: Lng roi ca L. monocytogenes

Vi khun L. monocytogenes sng rt dai v khc vi a s vi khun khc L.
monocytogenes c th tng trng chm trong t lnh nhit 4C.
Hin nay cc nh khoa hc cho rng con ng nhim L. monocytogenes ph bin nht
l qua thc phm.
Trong s by loi Listeria c bit n, ch c L. monocytogenes mi l tc 11
chng huyt thanh (srotypes) trong 90% trng hp bnh Listeriosis ngi u do
cc serotype 1a, 1b v 4b gy nn. Trong ba nhm va k trn, th 4b l serotype c hi
nht.
2.2.3.2. c im sinh ha
Ty tng loi hay tng kiu huyt thanh m Listeria. spp c cc biu hin sinh
ha khc nhau. c im sinh ha chung ca Listeria. spp c th hin nh sau.







Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 27

Bng 2.4: c im sinh ha ca mt s loi Listeria
Loi
Acid sn sinh t
-Hemolysis
1
Manitol Rhamnose Xylose c tnh
2

L. monocytogenes
+ - + - +
L. ivanovii
3

+ - - + +
L. innocua
- - V
4
- -
L. welshimeri
- - V
4
+ -
L. seeligeri
+ - - + -
L. grayi
5

- + V
4
- -
1
Th nghim trn mi trng thch mu cu.
2
Th nghim trn chut.
3
Nhng chng ln men ribose c phn thnh L. ivanovii subsp. Invanovii v khng
ln men nh L. ivanovii. Lomdiniesis.
V
4
: biotypes thay i
5
Bao gm hai loi ph L. grayi. Murrayi bin i nitrate v L. gayi subsp. Grayi khng
bin i nitrate

Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 28

2.2.4. Cu trc
2.2.4.1. Cu trc t bo

Hnh 2.9: Cu to t bo L. monocytogenes.

2.2..4.2. Cu trc phn t
Kch thc b gen ca loi L. monocytogenes khong 3,0 Mb
(Ganbank/EMBL_accession number AL591824). Nhng gen qui nh tnh gy c nm
trn b nhim sc th ca chng Listeria sn xut protein cho php to yu t iu ha
gip chng thch nghi vi iu kin mi trng khc nhau v iu kin c tnh gy
c.
Bng 2.5: B gen ca loi L. monocytogenes
L. monocytogenes
Kch thc nhim sc th (kb) 2.944.528
T l G+C (%) 39
T l G+C ca gen m ha protein 38
Tng s lng gen m ha protein 2.853
Lng
Nhn
Tin mao
Ribosomes
Mng t
bo
V t
bo
Bao v
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 29


Mt khc, cc loi nh L.innocua thiu gen qui nh cho mt protein to c t
nn khng th m ha cho protein ng vai tr quan trng cho vic xm nhp vo t bo
ch. iu ny c th gii thch c kh nng gy bnh gia cc loi khc nhau thuc
cng ging Listeria. C L. monocytogenes v L. ivanovii u c mt t hp gen qui nh
c tnh vi chiu di 8,2 Kb trn b gen, c iu ha bi yu t PrfA (Positive
regulatory factor A).T hp gen ny nm gi gen prs v gen ldh trn nhim sc th.
2.2.5. Yu t c lc

PrfA





Hnh 2.10: V tr t hp gen qui nh c tnh trong L. monocytogenes.

Phn ln t hp gen ny tham gia trong vic xm nhp vo t bo ch v tin
hnh chu k xm nhim bn trong cc t bo ca tc nhn gy bnh. T hp ny m ha
bao gm 6 gen plcB, hly, mpl, actA, plcB, prfA v c 3 khung c m X, Y, Z.
Trong gen prfA rt cn thit cho tnh c ca loi L. monocytogenes, c vai tr
nh yu t iu ha chnh nhm n nh hot ng ca t hp gen ny. Bn cnh ,
gen hly m ha protein to sn phm listeriolysin O (LLO), plcA m ha to
phosphatidylinositol-specific phospholipase C (PI-PLC), plcB m ha to
phosphatidulcholine-spefic phospholipase C (PC-PLC).
Tng t nh LLO, cc phospholipase C ny dung gii t bo ch bng cch to
mt l nh trn t bo v ph v lipid mng t bo. Vi khun sinh ra mt protease mpl
ph thuc Zn
+
c hot tnh nh mt vi ngoi c t.
Z
X plcB Y mpl actA
hly plcA prfA
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 30

Ngoi ra c mt vi gen c qui nh tnh gy c nhng t hp gen ni trn bao
gm inlA, inlB, inlC m ha ln lt cho internalin A, B v C. Cc gen ny ng vai tr
quan trng nhm ni b ha t bo ch do tc nhn gy bnh. Tuy nhin tt c cc gen
trong t hp gen gy c v tt c cc gen nm ngoi t hp gen ny vn hon ton ph
thuc vo hot ng ca yu t prfA.
Bng 2.6: Chc nng ca cc gen trong hot ng to c t ca L. monocytogenes.
Gen Chc nng
prfA Positive regulotory factor A
Hot ha cho qu trnh ly lan tronh t bo
ch ca vi khun.
plcA
Phosphatidylinositol-specific
phopholipase C
Dung gii mng khng bo ca th thc bo.
plcB
Phosphatidylcholine-specific
phospholipase C
Dung gii mng khng bo ca th thc bo.
hlyA Listeriolysin O Gii phng vi khun ra khi khng bo.
Mpl Zinc-dependent metalloprotease
Tham gia trong qu trnh hot ng ca
plcB.
actA Actin-polymerizing protein
Gip ly nhim t t bo ny sang t bo
khc.
inlA Internalin A Ni b ha t bo ch.
inlB Internalin B Ni b ha t bo ch.
2.2.5.1. Listeriolysin O
LLO l mt yu t c lc chnh c sinh ra bi L. monocytogenes gip cho L.
monocytogenes thot khi cc khng bo thc bo u tin. N l mt protein c trng
lng phn t 58,5 kDa c to thnh bi mt chui n polypeptide gm 529 aa.
Trnh t u N c c im ca trnh t du hiu in hnh vi khun Gram dng:
phn u c tnh i nc v mang in tch dng c theo sau bi 20 u k nc.
Trnh t ny m ha trc tip cho protein v c phn ct sau v tr lysine 25. Kt qu
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 31

l LLO c bt u t amino acid 26, l mt phn t protein di 504 aa v c trng
lng phn t 55,8 kDa.
LLO thuc v h c t gn cholesterol c hot bi nhm thiol c to ra bi
hu ht cc vi khun Gram dng. Mt s c t thuc h ny nh Streptolysin O t
Streptococcus pyogenes, pneumolysin O t Streptococcus pneumoniae, perfringolysin O
t Clostridium perfringens, cereolysin O t Bacillus cereus, alveolysin O t Bacillus
alvei Hot tnh ly gii ca cc c t ny c tng cng bi cc tc nhn c ch v
b km hm bi s oxy ha v biu hin khi c cholesterol hoc khng th khng
streptolysin O. Cytolysin c hot ha bi thiol c mt cystein n vng u C lm
cho chng d b bt hot bi s oxy ha mc d cystein ny c th c thay th bng
alanine m khng lm mt chc nng ly gii. Chng to nn nhng l khng lin tc vi
ng knh bn trong ln n 30 nm trn nhng mng cha cholesterol. C ch hot
ng ca chng da vo s tng tc vi cholesterol vi chc nng khng ch l v tr
gn kt m cn nh mt tc nhn lm thay i hot ng ca enzyme dn n qu trnh
oligomer ha ca khong 20 80 toxin monomer thnh cu trc vng hoc ging vng
cung.
LLO cho thy s tng tc k l vi cholesterol trong dung dch nh sau: protein
b bt hot bi cholesterol nhng, thay v ging cc c t khc, duy tr kh nng gn
trn mng. 1 HU (hemolytic unit) LLO c th bt hot 0,2 ng cholesterol trong khi cc
sterol khc nh epicholesterol v dehydroepiandrosterone l nhng cht c ch yu.
Cht oxy ha HgCl
2
v p chloromercuribenzoate bt hot c t nng 1 mM. S
c ch bi cc hp cht thy ngn b o ngc bi dithiothreitol 2 mM hoc cystein,
iodacetic acid 2 mM, iodacetamide 2 mM, 1 mM tosyllysine chloromethyl ketone v 1
mM tosylphenylalanine chloromethyl ketone khng c ch.
Hot tnh dung huyt ca LLO c lin quan n pH ca mi trng: n hot ng
cao nht pH 5,5 v gn nh b bt hot pH 7,0. Tri li, cc c t c hot ha
bng thiol c ph pH hot ng rng hn nh pH 6.0 i vi pneumolysin, pH 6.5 i
vi perfringolysin O v alveolysin, pH 7.0 i vi streptolysin O. S pht hin ny c
th gii thch vai tr ca LLO trong qu trnh xm nhim ca L. monocytogenes. Sau
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 32

qu trnh thc bo, mi trng acid ca khng bo cha t bo vi khun s hot ha
LLO. Chnh c t ny s xc tin qu trnh ly gii khng bo v t bo vi khun xm
nhp vo t bo cht trong khi , mi trng pH cao hn s lm gim hot tnh ca
LLO. S h hi ca mng t bo ch cng b ngn cn bi s phn hy nhanh ca LLO
trong t bo cht do s nhn bit ca mt trnh t ging PEST. Mt trnh t amino acid
giu Proline (P), glutamic acid (E), serine (S) v threonine (T) c gi l PEST v
protein ch phn hy. V u N ca LLO nm vng 19 aa (t 32 50) chng
minh c im ny. Mt iu th v khi loi b motif ging PEST ny khng ch nng
cao c lc ca LLO m cn sa i cc yu t c lc ca vi khun. L. monocytogenes
b t bin, thiu trnh t PEST LLO, c chng minh l vic thot ra khi khng bo
thc bo theo mt con ng rt kh khn. iu ny chng t rng vng ny cng rt
quan trng cho qu trnh ph hy mng.
2.2.5.2. Vai tr ca cc yu t c lc trong qu trnh xm nhim ca
L. monocytogenes
L. monocytogenes xm nhim vo t bo ca c th vt ch thng qua c ch
thc bo. Nu L. monocytogenes vn cn sng sau giai on u xm nhim ny, n
c c th vt ch tip nhn nh protein b mt c gi l internalin, ng ch l
internalin A (InlA), internalin B (InlB). InlA l protenin 88 kDa c m ha bi gen
inlA tng tc vi E Carherin to iu kin cho L. monocytogenes i vo t bo biu
m, InlB l protein 65 kDa c m ha bi gen inlB, nhn din Clq R (hoc Met) cho
php L. monocytogenes xm nhp vo trong nhiu loi t bo ca vt ch nh t bo
gan, nguyn si bo v cc t bo biu m.
Tc xm nhim ca t bo vt ch nhanh cho php L. monocytogenes trnh
khi s kim sot ca chc nng min dch. Sau khi xm nhim c vo t bo vt
ch, L. monocytogenes ch yu nm mng n ca khng bo, phn gii mng n s
cp ca khng bo, nh vo s kt hp ca hai phn t gy c: listeriolysin O (LLO) l
protein 58 kDa c m ha bi hly, to ra cc l trn mng t bo, hot gii c tnh
lu hunh l yu t cn thit cho tnh c ca Lm. Phosphatidylinositol-phpspholipase
C (PI-PLC) l protein 33 kDa c m ha bi gen plcA, kt hp vi
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 33

Phosphatidylcholine-phospholipase C (PC-PLC, protein 29 kDa c m ha bi plcB),
gip LLO phn gii mng n s cp ca khng bo.
Sau khi phn gii mng n s cp ca khng bo, L. monocytogenes c
phng thch vo cytosol, ti y chng pht trin v nhn ln, ni bo chng di chuyn
t t bo ny sang t bo khc, s di chuyn ny i hi phi c protein b mt khc
nh ActA (protein 67kDa c m ha bi gen ActA), ng sao chp vi PC PLC v
iu chnh s thnh lp ca protein cu thnh lng ui b phn c nhm a L.
monocytogenes vo mng t bo cht
Ti y, L. monocytogenes tr thnh dng v bao trong cu trc ging nh chn
gi dng si, xm nhim vo cc t bo cn k v phn gii mng i ca khng bo th
cp. khi mng i b phn gii l du hiu bt u 1 chu trnh xm nhim mi, n ph
thuc vo hot ng ca PC-PLC v Mpl (l metallprotease 60 kDa c m ha bi
gen mpl).
2.2.6. Con ng xm nhim

Hnh 2.11: Qu trnh xm nhim v tc hi ca L. monocytogenes.

Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 34

Sau khi xm nhim vo c th qua ng tiu ha, L. monocytogenes tip tc di
chuyn qua d dy ri i vo cc m rut non ca vt ch, bao gm cc t bo biu
m v cc i thc bo ca mng Peyer. Trong sut qu trnh di chuyn chng s khng
gy ra nhng thng tn m hc nu liu lng nhim khng ln. Trong dch
lympho hoc mu th L. monocytogenes di chuyn rt nhanh n hch bch huyt, l
lch v gan ri nhanh chng i su vo khng bo, hu ht vi khun xm nhp v ph
hy khong bo nh kt hp vi pH thp, cc enzyme thy phn v oxi ha trong khng
bo.

Listeriolysin dung gii Lan truyn t t bo ny sang t bo khc
i thc bo

hnh 2.12: Qu trnh xm nhim v ly lan ca Listeria monocytogenes

T khng bo chng s i vo t bo cht v nhn ln trong vng 30 pht sau khi
xm nhim, t 40-60 pht trong t bo gan v i thc bo. Qu trnh xm nhim vo
c quan hon thanh sau 5-7 ngy. Tuy nhin nu khng t c p ng min dch t
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 35

bo bch huyt CD8
+
v gamma, interferon gin tip hot ha i thc bo. Nu t bo
ca vt ch khng to ra p ng min dch th Listeria monocytogenes s di chuyn
n cc v tr khc trong c th nh no v nhau thai.
S pht tn ni bo v gian bo ca L. monocytogenes

Hnh 2.13: Cch xm nhim ni bo ca L. monocytogenes.
a)L. monocytogenes xm nhp vo t bo. b)Vi khun pht trin ni ti khng
bo. c,d). Mng ca khng bo bi ph v bi hai phospholipases: PicA, PicB v hnh
thnh cc c t listeriolysin O. Vi khun di chuyn vo trong t bo cht, ni chng
nhn ln v bt u tng hp actin. e)ui Actin cho php vi khun i vo mt t bo
ln cn bng cch hnh thnh ch nh ra trn mng huyt tng. f)Vi khun ph v t
bo thot ra ngoi v ko di chu k.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 36

Tm li s xm nhim ca L. monocytogenes c m t theo cc bc nht nh
sau:
S xm nhim v tip thu mm bnh
Thot khi th thc bo ca khng bo
Nhn ln trong t bo cht ca vt ch
nh hng di chuyn trong t bo cht
2.2.7. Kh nng gy bnh
2.2.7.1. Trn ngi
Bnh do Listeria gy ra l mt bnh him gp ngi nhng v cc triu chng
rt nguy him v gy t l cht kh cao do cn bnh ny nhn c s quan tm
ca cc cp chnh quyn a phng v vn sc khe. Phn ln ngi b nhim u
c cc du hiu cn lm sng nh. Thng thng i vi ngi khe mnh, s tiu th
thc phm b nhim l nguyn nhn gy ra vim d dy rut, triu chng st. Tri li,
L. monocytogenes l tc nhn gy cht c bit l tr em di 1 thng tui, ph n
mang thai, nhng ngi nhn m cy ghp v nhng bnh nhn c h min dch km.
LM hng s kch thch vo h thn kinh trung tm v c cc du hiu lm sng l vim
mng no v hnh thnh nn p xe. S nhim trng l mt dng bnh khc do listeria
gy ra tc ng n cc bnh nhn mang bnh v min dch. Ngoi cc bnh ni chung
ny, vi khun ny cng gy nhim nh v nh vim mng kt, nhim trn da, bnh ca
hch bch huyt. ph n mang thai, khi ngi m b nhim Listeria th khng c triu
chng r rng hoc c nhng triu chng ging nh b cm cm trong khi kt qu bo
thai v thai nhi b nh hng nghim trng hn bao gm sy thai, cht non, vim mng
no tr s sinh hay nhim trng.
2.2.7.2. Trn ng vt
Trong s cc ng vt, bnh do listeria tc ng chuyn bit trn gia sc, cu v
d vi thi gian bnh t 2 n 6 tun. ng vt b nhim Listeria khi n phi thc n
nhim, qua ng h hp hoc do tip xc trc tip. S pht bnh xy ra km theo s
suy gim min dch i vi tc nhn gy bnh do cc yu t gy stress (mang thai, s
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 37

ng c, s vn chuyn, iu kin mi trng km). ng vt b nhim bnh do
listeria thng st, bing n, suy nhc v mt phng hng
S biu hin bnh c th hnh thnh nn cc du hiu lm sng nh sau:
Thn kinh (bnh quay vng) nh bnh vim no v vim mng no. Du
hiu lm sng bao gm s n n, u quay v mt bn, i vng vng, hc u vo vt
rn. Nhng s biu hin ny do s hnh thnh cc vi p xe trong ty xng, xng
sng v trong tiu no.
Ni tng nh nhim trng mu. Ban u n thng xy ra chim, bo thai
hoc ng vt khong 1 thng tui. S nhim ny xy ra gy hoi t gan vi cc vng
tn thng mu trng xm v c th lan n l lch.
S sinh sn nh sy thai, non. iu ny rt hay xy ra ng vt n c.
Vi khun xm nhim vo t cung thng qua mch mu v s sy thai thng xy ra t 5
- 12 ngy sau khi vi khun tng trng trong nhau.
S nhim v nh chng vim v. Triu chng ny thng do s nhim
cn lm sng m khng c bng chng sng v. Sa do sinh vt tit ra lm tng ri ro
nhim trong sn phm sa.
Vim mng mt l triu chng thng t gp.
2.2.8. Phn b
Listeria monocytogenes phn b rng ri trong mi trng t, ct, nc. C th
truyn bnh qua ng ming v phn, v phn lp c t tht nu cha chn, gia
cm, sa ti cha thanh trng, phomat v hi sn.
L. monocytogenes l vi khun c mt khp ni trong rau c c th nhim khun
t t hoc t phn bn. Sc vt nng tri c th nhim khun m khng c triu
chng g v thc phm t ng vt nh tht, tht gia cm v cc sn phm sa c th b
nhim khun.
Trong khi vi khun c th tm thy cc loi thc phm ti sng nh tht cha
nu chn, rau c qu, sa cha tit trng v thc phm t sa ti, thc phm ch
bin sn nh rau qu ct lt c th nhim khun sau quy trnh sn xut. Cc thc phm
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 38

khc nh c th nhim khun L. monocytogenes khi thc phm thng mi lm nhim
khun ny trn b mt bp v thc phm khc hoc thng qua nc p qu ng hp.
Vi khun ny rt bn. Chng khng nhit (mc du nhit khng cao), mui,
nitrite v acid, sng st iu kin lnh ng v thm ch c th sinh si pht trin
chm chp trong t lnh. Do , con ngi c nguy c nhim khun cc k cao lu
cc loi thc phm cha nu chn k trc khi n c th c rt nhiu L. monocytogenes.
Theo S gim st v an ton thc phm thuc B nng nghip, nhng trng hp
bnh listeriosis M u lin quan n thc phm xc xch nh xng khi, tht hp ti
ngon, pat lnh, xc xch , pho pht mm ca Php, pho mt mm kiu Mhic, tm,
b, rau qu ti v sa cha tit trng. Trn dch bnh bng pht c th lin quan n s
hn tm cua, c ti, thy sn xng khi, li heo, kem, x lch go, x lch ci bp v
pat tht.
Vi khun c th nhim vo chng ta qua cc vt dng nh bp nh dao, tht bn
hoc t tay b nhim trng.
Nu nng thc phm v hp kh trng sa u dit c vi khun. Tuy nhin,
i vi mt s thc n lm sn (ready to eat products) nh tht g, cua v tht ngui nh
hot dog, deli meats, luncheon meats, v.vchng cng c th b nhim vo sau giai on
nu nng v trc khi c cho vo bao.
Chng ta cng c th b nhim khun L. monocytogenes nu c s tip xc trc
tip vi th c mang vi khun ny.
2.2.9. Tnh hnh nhim Listeria monocytogenes trong sn phm thy sn
trn th gii v Vit Nam hin nay
T l nhim bnh thanh nin khe mnh l rt thp, khong 0.7 trng hp trn
100.000 ngi. Tuy nhin, kh nng nhim tr em thng cao hn khong trn 10
trng hp trn 100.000 ngi v ngi gi t l ny l 1.4 trng hp trn 100.000
ngi. Ph n mang thai d dng b nhim bnh gp 17 ln so vi thanh nin khe mnh
2.2.9.1. Tnh hnh th gii
Nhiu nc trn th gii nh M, Thy S, nht l Php, t l nhim vi khun
ny trong thc phm nh sa ti v cc sn phm ch bin t sa, tht c ti sng v
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 39

cc sn phm ca tht c, rau xanh ... l rt cao. Nhim trng do Listeria monocytogenes
cng xy ra tri rc Chu u, Chu M v Chu Phi cng nh trong khu vc Chu
Ti M c khong 76 triu trng hp b ng c thc phm hng nm, trong
t l nhim do Listeria ch khong 2.5000 trng hp nhng c 500 trng hp t vong.
Mc d con s cc ca nhim bnh thp nhng t l cht cao chim t 20 30 % cc ca
nhim bt chp c s dng thuc khng vi sinh, cho thy mc nguy him ca s c
mt cc loi Listeria trong thc phm.t l t vong cao hn ng k so vi cc tc nhn
gy ng c thc phm khc nh Escherichia coli O157:H7 (E.coli),
Campylobacter.spp v Samonella.
Bng 2.7: So snh t l t vong gy ra gia L. monocytogenes v cc tc nhn
gy bnh khc
Tc nhn gy bnh
T l
t vong (%)
T l
nhp vin (%)
Tng s
ca nhim bnh
Campylobacter.spp <1 17.3 gn 1.9 triu
E. coli O157:H7 <1 3.0 gn 62.500 triu
Samonella <1 25.6 gn 1.3 triu
L . monocytogenes 20.0 3.8 2.500

Liateria c xem l vn ghim trng nhim vo cc sn phm tht v tht gia
cm vo nhng nm 1980. vo nhng nm 1990, B Nng Nghip M cho bit c mt
trn dch bnh nguyn nhn t bnh m kp xc xch nng dn n t nht 101 ngi
bnh, khin 15 ngi ln t vong v 6 thai nhi sinh non.
Nm 1999, loi L. monocytogenes cc c tin trin bo ng cc vin chc y
t v h buc nhng nh sn xut thc phm gii quyt vn . Ngi c nguy c cao
c th mc bnh ny sau khi n thc phm thm ch nhim vi vi khun
Bnh dch Listeriosis nm 1992 Php xut pht t li heo, lan truyn qua dao
xt tht, lm 66 ngi cht, 22 ph n sy thai. Bnh dch nm 1993 nh hn, tm thy
vi khun trong nhng hp ch heo v ch c vi ca t vong.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 40

Gn y nht, 12/10/2002, dch Listeriosis ti cac ban Ty Bc nc M c 23
ngi cht v 120 ngi ng c phi vo bnh vin, nguuyn nhn l tht g ca hng
Pilgrims Pride nhim Listeria monocytogenes. Tht g khng ch gy ng c Listeria
m cn nhiu vi khun khc. Nc di g v rut g c sn nhng vi khun gy bnh
nh Salmonella, Shigella, Campylobacter. Nhng khun ny khng gy bnh g m
gy bnh ngi (au bng, tiu chy, thng hn).
Bng 2.8: T l pht hin s c mt ca L. monocytogenes trong tht v tht gia
sc ca cc nghin cu mt s quc gia trn th gii
Quc gia T l pht hin % S lng mu nhim
Ty Ban Nha 64 28
Norway 61 71
Anh 60 61
M 23 6
Belgium v Php 10 15 48
Ireland 59 91

2.2.9.2. Tnh hnh Vit Nam
Nm 1996, trong s 215 mu thc phm cc loi gm sa chua, hi sn ng
lnh, tht ti sng, rau xanh v thc phm ch bin dng ngay, Vin Pasteur TP HCM
phn lp c 2 dng L . monocytogenes, chim t l 0.93%. n nm 1999, vin65
V Sinh Y T Cng Cng cng phn lp c 2 dng L .monocytogenes t 20 mu
thc phm, chim t l 10%.
Nm 2006, theo tng kt ca Phng Kim Nghim Ha L Vi Sinh thc phm
vin Pastuer TPHCM, cc mu hi sn ng lnh c t l nhim L. monocytogenes
23/138 mu, chim 23.9%. Nh vy t l xut hin ca chng vi khun ny cng c
khuynh hng gia tng theo thi gian nc ta v thng c tm thy cc mu thc
phm ng lnh.
Theo Quyt nh s 2670 ca B Nng Nghip v Pht Trin nng thn cp ngy
29/8/2008 v vic Cng b danh mc cc ch tiu ch nh kim tra i vi l6 hng
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 41

thy sn, ch tiu L. monocytogenes quy nh gii hn pht hin l 0cfu/25g i vi
hu ht cc loi sn phm thy sn.
Bng 2.9: Gii hn pht hin cho php i vi L. monocytogenes trong sn phm
thy hi sn.
Loi sn phm Gii hn cho php i vi L. monocytogenes
Thy sn n lin 0 cfu/25g
Thy sn ng lnh (bao gm
nhuyn th), khng cn nu trc
khi n
0 cfu/25g
C ng lnh (nguyn con, fillet,
ct khc...)
0 cfu/25g

2.3. Cc phng php pht hin Listeria monocytogenes
2.3.1. Phng php truyn thng
Thng quy k thut nh lng listeria monocytogenes trong thc phm.
2.3.1.1. Nguyn l phng php
S dng phng php cy m Listeria monocytogenes trn mi trng nui cy
chuyn bit sau khi m nhit 37
0
C trong 48 gi. Chn cc khun lc nghi ng
xc nh Listeria monocytogenes bng cc th nghim sinh vt ho hc.
2.3.1.2. Phm vi p dng
Pht hin s nhim do Listeria monocytogenes trong thc phm v sn phm
sa.
2.3.1.3. Dng c, mi trng v thuc th
Dng c, thit b:
1. 0,1g mu
2. Giy bc dng c thy tinh
3. Bnh tam gic 500 ml
4. ng ln men (Durham)
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 42

5. Cc loi bt nh du
6. T m 30 - 35
0
C
7. Du ngm
8. Que cy vng
9. Que cy thng
10. Knh hin vi
11. Mu ngm du (quan st th knh 100x)
12. a Petri
13. Pipettes, 25, 10, v 1 ml
14. ng nghim c np y
15. My xay hoc ti nghin mu
16. Cc dng c, thit b c v trng hoc s dng mt ln
Mi trng, thuc th:
1. Acid acetic, 5 N
2. Acriflavine monohydrochloride
3. Agar
4. N-(1-naphthyl) ethylene diamine
5. -Naphthol tinh khit
6. Thch mu (Unipath)
7. Cycloheximide
8. Natamycin (pimaricin)
9. Mu cu
10. Ethanol tuyt i
11. Khng th hunh quang (FA) m (Difco)
12. Glycine anhydride
13. B nhum gram
14. Hydrogen peroxide 3% cho th nghim catalase
15. KOH 40% (R65)
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 43

16. Huyt thanh cha Listeria (Difco)
17. Lithium chloride-phenylethanol-moxalactam (LPM) agar (M81) cng
esculin v st (M82)
18. Nalidixic acid (mui sodium)
19. Nitrate trung bnh (M108) v thuc th pht hin nitrat (R48)
20. Mi trng dinh dng (M114)
21. Nc mui sinh l 0,85% (R63)
22. Dch ng ln men (M130), cho cc th nghim 0,5% dextrose,
esculin, maltose, rhamnose, mannitol, v xylose
23. SIM trung bnh (Becton Dickinson, h thng Vi sinh vt, M137) hoc
nhu ng th nghim trung bnh (MTM, Difco) (M103)
24. Sulfanilic axit tinh khit (R48)
25. Trypticase u nnh agar vi chit xut nm men 0,6% (TSAye)
(M153)
26. Trypticase canh u nnh vi chit xut nm men 0,6% (TSBye)
(M157)
27. Oxford trung bnh (OXA) (M118)
28. Canh m tng sinh Listeria (M52)
29. PALCAM agar (M118a)
30. Carageenan (Sigma loi II)
31. BCM agar (M17a)
32. MOX agar (M103a)
33. ALOA agar (M10a)
34. Thch to mu cho Listeria (M40b)
35. Mi trng di ng ca Listeria (M131a)
36. mu Listeria (M40a)
37. canh v thch Tryptose (Difco) (M167)

Lu : cc mi trng thay th c th c s dng khi cc sn phm l tng ng.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 44

2.3.1.4. Chun b mi trng v mu th
Chun b mi trng
Mi trng nui cy, canh thang, nc pha long v mi trng sinh vt ho hc
c iu ch theo cng thc. Cc mi trng c ng sn vo bnh cu, bnh nn,
ng nghim v c hp tit trng (110
0
C/30 pht hoc 121
0
C/15 pht ).
Chun b mu v dung dch mu th:
Chun b mu
Mu thc phm c ct nh hoc xay nhuyn bng my trong iu kin v
trng cho ti khi c th ng nht.
Chun b dung dch mu th 10
-1
:
Cn chnh xc 25g thc phm c chun b (hoc ht 25 ml thc phm lng),
cho vo erlen cha sn 225 ml nc pepton. Lc u 2-3 pht, thu c dung dch mu
th 10
-1
.
Chun b dung dch mu th 10
-2
, 10
-3
, 10
-4
...
Ht chnh xc1ml dung dch mu th 10
1
cho sang ng nghim cha sn 9ml
nc pepton. Lc u trong 2-3 pht thu c dung dch 10
-2
. Tip tc lm tng t
nh vy ta thu c cc dung dch mu th tng ng 10
-3
, 10
-4
,10
-5
.
2.3.1.5. Phng php tin hnh
Thu mu v tng sinh
1. Bo qun mu: Mu c bo qun 4
0
C, trong qu trnh bo qun v vn
chuyn mu th L. monocytogenes vn pht trin c, mc d rt chm nhit ny.
Tuy nhin, nu mu c ng lnh, th khng nn x cho n khi phn tch.
2. Mu phc hp: Thng thng, phc hp mu c chun b sao cho mu i
din c b mt v bn trong ca thc phm. Cc mu ly i din trn mt thc
phm c trn li v cho vo 250 ml dung dch m tng sinh Listeria c cha sodium
pyruvate, khng c cha mi trng chn lc. Cc mu khc cng thc hin tng t.
Thng thng 50g ca hn hp pha trn c pha vi 200 ml dung dch m trc khi
tng sinh. Mu c bo qun trong khong nhit t 0
0
C 5
0
C.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 45

3. Mu khng phc hp: Nu mu khng yu cu phc hp, n gin chn 25g
mu thc phm pha vo 200 ml dung dch m v thc hin cc bc tng sinh tip
theo.
4. Tin tng sinh v tng sinh: mu trong 4 gi 30
0
C, thm cc mi trng
chn lc v tip tc thm 48 gi 30
0
C. Nu cycloheximide khng c sn, th c th
thay th bng pimaricin (natamycin) nng 25 mg/l. Natamycin an ton v d s
dng hn cycloheximide.
5. Lit k cc mu dng tnh vi L. monocytogenes: Dng mt s phng php
xc nh nhanh lit k cc mu dng tnh vi L. monocytogenes, sau cc mu
tip tc c tng sinh v s lng L. monocytogenes c trong thc phm l rt thp
(khong 1 cfu/25g mu).
Cc bc xc nh
1. Chn mu nghi ng c cha L. monocytogenes. C th quan st vi nh sng
ca cc thit b chiu sng.
2. mu 30
0
C, ngm du, quan st di knh hin vi kim mc pht
trin. Chn mt mu vi tc tng trng t yu cu xc nh.
3. Kim tra sinh ha ca cc loi Listeria l catalase dng tnh.
4. Tin hnh nhum Gram. L. monocytogenes l vi khun Gram dng.
5. Th nghim ln men ng. Cc mu dng tnh c gi li xc nh cc
bc tip theo.
6. Th ngim vi thch mu cu: mt s loi L. monocytogenes lm tan thch
mu. L. monocytogenes c cy vo thch mu v trong 24 48 gi 35
0
C. C gng
cy L. monocytogenes vo lp thch sao cho gn n lp di cng ca thch mu.
7. Xc nh cc loi Listeria thng qua hin tng tan huyt: L.monocytogenes
to vng lm, L.seeligeri to hi, L.innocua cho thy khng c tn huyt, trong khi L.
ivanovii to ra mt vng cng c xc nh r rng xung quanh. Tn huyt c xc
nh d dng hn khi lp thch mu mng hn 5mm.
8.Th nghim Nitrate. 35
0
C trong 5 ngy. Cho vo 0,2 ml thuc th A, tip
theo l 0,2 ml thuc th B (R48) Mt mu sc -tm cho thy s hin din ca nitrit.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 46

9. Xc nh kh nng di ng vi thch SIM: L. monocytogenes dng tnh vi
hnh anh di ng hnh d.
10. Th nghim ln men tao acid vi: dextrose, esculin, maltose, rhamnose,
mannitol, v xylose. 7 ngy 35
0
C. Listeria spp thng dng tnh cho sn phm
acid khng c gas. Tt c cc loi Listeria u dng tnh vi dextrose, esculin, v
maltose. cc th nghim esculin c th b qua.
Th nghim Methyl Red v Voges Proskauer (MR-VP):
T canh thang TSB-YE cy vo canh thang Clark-Lubs .
m : 35
o
C/48h.
Ly 1ml vo ng v trng v nh thuc th: 0,2ml KOH 40% v 0,6 ml a
Napton.
nhit thng trong 1h. c kt qu. Nu dng tnh c mu .
m canh thang cn li thm 2 ngy. Nh Metyl 0,5% trong cn 60
0
.
c kt qu: Nu dng tnh c mu .
Th nghim CAMP Test
Ria chng Staphylococcus aureus NCTC 1803 v Rhodococcus equi
NCTC 1621 to thnh nhng ng ngang qua a thch mu cu 7%.
Ria khun lc kim tra gia, vung gc vi Staphylococcus aureus v
Rhodococcus equi.
m 37
o
C/24h.
c kt qu: Nu thy vng tan mu tin v pha ng cy chng
S.aureus l Listeria monocytogenes.
Ch : ng cy chng kim tra khng c chm vo chng Staphylococcus aureus
v Rhodococcus equi v cch chng khong :1-2mm.
B mt thch mu phi kh.
Tiu chun xc nh Listeria monocytogenes trong thc phm:
Trc khun, Gram ( + ) Catalaza ( + ) MR ( + ) VP ( + )
Xylose ( - ), Rhamnose (+), Manitol (-), Di ng (+) 20
0
C-25
0
C
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 47

Nu cn : Ngng kt vi cc khng huyt thanh : H v O
CAM Test:
+ Staphylococcus aureus (+)
+ Rhodococcus equi ( -)
S nh lng L. monocytogenes.
m: p ngc a thch, m 37
O
C 48 gi.
Chn a thch: Chn a c t hn 300 khun lc m.
Tng sinh thun nht.
Xc nh tnh cht sinh vt, ho hc nh bng sau:
Bng 2.10: Tnh cht sinh vt ha hc ca L. monocytogenes.
STT Phn ng sinh ha Biu hin ca L. monocytogenes
1 Nhum gram + (Gram dng)
2 Catalase +
3 Kh nng di ng (20
0
-25
0
C) + (Hnh d)
4 Ln men Xylose -
5 Ln men Rhamnose +
6 Phn ng KOH -
7 Phn ng CAMP +
8 Kh nng tan huyt +
9 Manitol -
10 MR +
11 VP +
12 Sinh hi -

2.3.2. Cc phng php hin i
Pht hin vi sinh vt bng phng php nui cy c xy dng v pht trin
trong thi gian di, c nhiu nc cng nhn v chng tr thnh nhng phng
php tiu chun. Tuy vy nhc im ln nht ca phng php nui cy l tn nhiu
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 48

thi gian cho kt qu chm, mt nhiu cng sc, cng knh v ngy cng t ra khng
p ng c cc yu cu phn tch phc v nhu cu thc t hin nay. khc phc
nhng nhc im trn ca phng php nui cy, nhiu phng php nhanh v t
ng c hnh thnh v pht trin da trn nhiu nguyn tc sinh hc v vi sinh vt
hc khc nhau. Cc phng php mi ny nhm mc ch rt ngn thi gian phn tch,
gim thiu s phc tp trong thao tc, d dng thc hin v c nhy cao v chnh
xc cao.
Cc phng php nhanh v t ng ha trong phn tch vi sinh vt thc phm
u da trn nguyn tc ca vi sinh vt hc, sinh ha hc, ha l, min dch hc, sinh
hc phn t pht hin v nh lng vi sinh vt mc tiu hay sn phm c hi ca
chng trong thc phm, mi trng. Tt c cc yu cu k thut lin quan n vic thu
v chun b mu phn tch u c nghin cu, ci tin theo hng t ng ha, n
gin ha cho kt qu nhanh v chnh xc.
2.3.2.1. Phng php Elisa (Enzyme Linked ImmunoSorbent Assay)
Nhng tin b khoa hc trong nhng nm gn y thc y s pht trin ca
cc k thut chn on bng huyt thanh. Cc phng php ny rt hiu qu trong vic
chn on nhanh v chnh xc cc vi sinh vt gy bnh. Ngy nay phng php ny cn
c pht trin xc nh cc cht c hi trong mi trng nh c t, d lng
khng sinh Nguyn tc ca phng php Elisa da trn phn ng kt hp gia khng
nguyn vi mt khng th c hiu. Phn ng min dch xy ra c pht hin bng
cch s dng nhng khng th c nh du (bng cch nhum pht hunh quang,
ng v phng x, hay enzyme).
Phng php ELISA (Enzyme Linked ImmunoSorbent Assay) l phng php
hp ph min dch lin kt enzyme Nguyn tc k thut Elisa l s dng khng th n
dng (khng th s cp) ph bn ngoi nhng ging nh nhm mc ch thu gi nhng
khng nguyn mc tiu. Nhng khng nguyn thu gi c pht hin bng cch s
dng khng th th hai c gn vi enzyme pht tn hiu (thng l horseradish
peroxidase hay alkaline phosphatase). Khi cho vo hn hp phn ng mt c cht c
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 49

hiu ca enzyme, phn ng xy ra v to cc sn phm lm i mu phn ng. V vy
chng ta c th pht hin c s hin din ca khng nguyn.

Hnh 2.14: C ch phn ng ELISA
Quy trnh thc hin phn tch ELISA c cc bc chnh nh sau:
t mu vo trong cc ging c cha khng th s cp, cho khng th th cp
lin kt vi enzyme to mu vo to sandwich, ra loi b cc khng th mang
enzyme khng tham gia phn ng cho c cht to mu vi enzyme lin kt trong hn
hp, o cng mu to thnh xc nh lng khng nguyn trong mu.
Hin nay Elisa c s dng rng ri phn tch Salmonella, E.coli gy
bnh, Listeria, c t Staphylococcus, thuc tr su, d lng khng sinh i vi vi
sinh vt, phng php Elisa c th s dng pht hin v nh lng vi sinh vt trong
thc phm sau vi gi tng sinh nhm tng nhy ca phng php.
2.3.2.2. Phng php PCR (Polymerase Chain Reaction)
Tt c cc mc tiu khuch i c pht hin bng mt s c im c bn. S
dng quy trnh cng ngh enzyme trong mt enzyme n hoc enzyme tng hp
nhiu bn sao ca cc mc tiu axit nucleic. Tuy nhin, phn ng chui polymerase
(PCR) l phn ng u tin v vn l k thut c p dng rng ri nht. PCR l mt
phng php n gin nhanh chng khuch i chui DNA mc tiu. mt DNA
polymerase nhit c kh nng tng hp DNA c th, v si kp DNA chc nng nh
mt mu khun thc hin phn ng DNA polymerase. Qu trnh PCR bt u
nhit cao ( 94
0
C) bin tnh v tho xon DNA si kp thnh DNA si n, tip
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 50

theo l nhit tng i thp (54
0
C) kch hot qu trnh bt cp ca cc nucleotide
v mch khun, v sau l 72
0
C cho php sao chp DNA polymerase ca mu.
Ton b qu trnh c lp i lp li 25 - 30 chu k mt bn sao ca mu DNA c
th bin thnh hng t bn sao trong vng 3 - 4 gi.
Gel in di thng c s dng pht hin cc sn phm khuch i. Khi ht
mi xunh quanh DNA khun, khi lng v kch thc ca gel ch c th c d
on v pht hin vi mt vt DNA (Wang et al., 1993; Manzano et al., 2000; Volokhov
et al, 2002.). Bng cch xc nh s khc bit trong phn t gen 16S v 23S rRNA,
vng gel m, hly, inlA, inlB, IAP v gen khc ca L. monocytogenes c nhanh
chng v phn bit chnh xc gi Listeria khc loi v vi khun thng thng. PCR
kho nghim a thnh phn c s dng khuych i mt cch chn lc gene
IAP v to thun li cho vic xc nh s khc bit ca tt c su loi Listeria trong mt
th nghim n (Bubert et al., 1999). Tuy nhin, nn cn thn trng xc nh kch
thc ca sn phm khuch i, c th thy s khc bit kch thc gia cc loi
Listeria. Nh vy, xc nh gen mc tiu cho mt loi Listeria duy nht c th c tt
hn, v n cung cp mt biu hin c lp xc nhn ca loi (Gilot v ni dung, nm
2002; Liu et al., 2003b, 2004a, 2004b, 2004c, 2005b). Nu ng hin din ca mt s
loi vi khun Listeria lm phc tp vic xc nh cc L. monocytogenes, th kho
nghim a thnh phn l rt hu ch nhn dng v c tnh ca vi khun phn lp.
Vic s dng cc on mi PCR nhm c th, cung cp cng c b sung xc nh
L. monocytogenes (Jinneman v Hill, 2001; Borucki v Call, 2003; Doumith et al. ,
2004a).
Tt c cc DNA polymerase u cn nhng mi chuyn bit tng hp mch
DNA mi t mch khun. Mch khun thng l nhng trnh t DNA ca gen nh c
trng cho loi vi sinh vt mc tiu hoc cc gen quy nh vic tng hp mt loi c t
chuyn bit. Mi l nhng on DNA ngn c kh nng bt cp b sung vi mt u
ca mch khun v nh hot ng ca DNA polymerase on mi ny c ni di
hnh thnh mch mi. Khi cung cp hai ngun mi chuyn bit bt cp b sung vi hai
u ca trnh t DNA, vi iu kin DNA polymerase hot ng trong phn ng PCR,
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 51

mt on DNA nm gia hai u mi s c to nn. Nh vy khuch i trnh t
DNA xc nh, ta c nhng thng tin ti thiu v trnh t to cac mi b sung
chuyn nghip. Cc mi ny gm mt mi xui (sens primer) v mt mi ngc
(antisens primer) so vi chiu phin m ca gen.
Phn ng PCR l mt chui nhiu chu k ni tip nhau. Mi chu k gm 3 bc:
bin tnh (denaturation), lai (anealation), tng hp (elongation). Phn tch sn phm
khuch i bng s in di trn gel agarose. c trng ca phn ng khuch i i vi
tng vi sinh vt mc tiu thng qua kch thc ca sn phm khuch i.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 52

S phn ng PCR:







Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 53

K thut PCR (Polymerase Chain Reaction) vi cp mi LM-F LM-R:
Chng vi sinh vt: L. monocytogenes
Chun b th nghim:
Trnh t gen hlyA ca cc chng L. monocytogenes c cng b trn
ngn hng gen.
Phn mm BLAST dung xc nh trnh t gen ch hlyA ca cc L.
monocytogenes. (Kanamyxin, Xgal, Eco RI, b kit tch dng Topo TA Cloning Kit, b
kit tch v tinh sch plasmid QIA prep Spin Miniprep Test Kit),kit xc nh trnh t
BigDye Terminator v3.1
Cp mi LM-F v LM-R c thit k bao gm 20 bp/mi cho sn
phm PCR 468 bp.
Ho cht s dng cho tch chit DNA tng s, ho cht cho in di trn
gel agarose, thang DNA chun 100 bp v 1000bp .Cc ho cht khc tinh khit
phn tch.
Phng php tin hnh th nghim : Mi cho phn ng PCR
Da trn cc trnh t gen hlyA ca cc chng L. monocytogenes c cng b
trn ngn hng gen, cp mi LM-F v LM-R c thit k bao gm 20 bp/mi cho sn
phm PCR 468 bp .
Xc nh tnh c hiu ca mi
Kh nng bt cp chnh xc ca mi c khng nh bng cch phn tch trnh
t sn phm PCR theo phng php ca Sanger v cng s v so snh vi trnh t cc
gen ch hlyA ca cc L. monocytogenes theo phn mm BLAST. Tnh c hiu ca mi
c kho st bng cch kim tra kh nng bt cp ca mi thit k vi cc khun DNA
ca vi khun L. monocytogenes v khng phi L. monocytogenes trong phn ng PCR.
Phn ng PCR
DNA khun t L. monocytogenes c thu nhn cho phn ng PCR bng cch
tch v lm sch DNA vi chloroform hoc x l nhit t bo 100
0
C trong 10 pht.
Ti u ho iu kin PCR v nng Mg
2+
(1,5 - 4,0 mM), nng mi (0,1 - 0,5
pmol/mi) v nng dNTPs (100 - 250 mM/mi loi). Phn ng PCR c thc hin
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 54

vi th tch hn hp 25 ml vi chu trnh nhit gm 94
0
C - 3 pht, (94
0
C 1 pht, 60
0
C
1pht, 72
0
C 1 pht) x 30 trnh, 72
0
C- 5 pht v 4
0
C - 5 pht. nh gi kt qu phn
ng PCR da trn phn tch sn phm PCR trn gel agarose 1% vi 3 ml sn phm.
Xc nh nhy ca phng php
Mi trng nui qua m ca L. monocytogenes trn mi trng LEB c nh
lng, pha long ti cc mt thch hp. Ly tm thu sinh khi ca 1 ml canh trng
ha tan trong 100 ml TE, x l nhit thu DNA cho phn ng PCR. 3ml dch t bo
qua x l nhit c s dng lm khun cho phn ng PCR. nh gi kt qu phn ng
PCR nh phn tch sn phm bng in di trn agarose 1%.
DNA b gen
DNA b gen t L. monocytogenes c tch v lm sch dng lm khun cho
phn ng PCR khuych i on gen mc tiu hlyA vi cp mi LM-F v LM-R, Kt
qu phn ng PCR vi khun DNA tinh sch cho thy mt bng DNA c kch thc
nm trong khong kch thc sn phm PCR thit k.Vi mc ch thu nhn DNA bn
mu cho phn ng PCR mt cch n gin hn, huyn ph L.monocytogenes c x l
nhit 100
0
C trong 10 pht ph v t bo, gii phng DNA, ly tm 10000 vng/pht
x 10 pht, thu dch ly tm cha DNA lm khun cho phn ng PCR.
Kim tra tnh c hiu ca cp mi LM-F, LM-R.
khng nh kh nng bt cp chnh xc ca cp mi LM-F, LM-R vi gen
hlyA ca L. monocytogenes, trnh t sn phm PCR ca cp mi ny c xc nh v
so snh vi kt qu x l plasmid trnh t gen ch hlyA ca cc L. monocytogenes. Sn
phm PCR sau khi c khuch i bng cp mi LM-F,LM-R c gn trc tip vo
vect pCR4 TOPO. DNA plasmid cha sn phm PCR c tch dng trong E.coli
DH5a. Tin hnh tch chit v tinh sch plasmid ti t hp (s dng b kit QIA prep
Spin Miniprep Test Kit), kim tra sn phm trn agarose 1%. Kt qu in di cho thy
cc plasmid ca khun lc trng c kch thc ln hn so vi i chng .
Sn phm PCR gn trong plasmid c kim tra bng cch ct vi EcoRI. Ti t
hp vi EcoRI cho thy on DNA chn vo plasmid c kch thc tng ng vi kch
thc sn phm PCR. Trnh t sn phm PCR c xc nh bng thit b xc nh trnh
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 55

t t ng ABI 3100 Avant. Trnh t ny c so snh vi trnh t hlyA ca ccL.
monocytogenes c cng b. Kt qu so snh cho thy sn phm PCR khuych i
bng cp mi LM-F, LM-R c kch thc 468 bp, t tng ng trnh t >98 % vi
trnh t hl yA ca 53 chng L. Monocytogenes cng b. Nh vy, cp mi LM-F, LM-R
thit k cho php nhn chnh xc on gen hlyA ca L. monocytogenes.
Xc nh iu kin ti u cho PCR.
Nng Mg
2+
:
Phn ng PCR c thc hin cc nng cui cng ca Mg
2+
t 1,5 mM n
4,0mM.
Nng dNTPs:
Phn ng PCR c thc hin vi nng dNTPs t 100 mM n 250 mM.
Nng mi
PCR c thc hin ti cc nng mi khc nhau t 0,1 n 0,5 pmol.
Dch t bo c xc nh mt bng phng php m khun lc. Pha long
dch vi khun ti cc nng khc nhau , x l nhit cc dch huyn ph vi khun. 3ml
dch sinh khi vi khun sau khi x l nhit dng lm khun cho phn ng PCR cho thy
phn ng PCR cho kt qu dng tnh vi cc mu c mt t bo 6x10
2
/phn ng
tng ng vi mt t bo t 2.10
4
CFU/ml.
Nh vy, phng php PCR pht hin L. Monocytogenes c nhy tng
ng vi kt thng mi BAX
TM
ca Qualicon. Inc., (10
4
CFU/ ml) nhng nh hn ca
Listeria monocytogenes LightCycler ca Roche (10
3
CFU/ ml). Cp mi LM-F, LM-R
thit k c hiu vi L. monocytogenes, cho php pht hin vi khun gy bnh L.
monocytogenes mt 6.10
2
CFU/phn ng (2. 10
4
CFU/ml).
B kit PCR:Vi nhy rt cao vi cc vi khun, c trong 25g mu thc
phm c mt con vi khun cn tm th PCR s pht hin ra chng. iu c bit l cc
b kt khng ch gi tn mt loi vi sinh vt gy ng c thc phm m chng c th
pht hin ra 12 loi vi khun khc nhau nh: e.coli, e. coli 0157:h7, salmonella spp,
shigella spp, nm mc... Ty thuc vo loi thc phm v s loi ch tiu vi khun gy
bnh cn kim sot i vi mi loi thc phm, cc quy trnh v b Kit PCR cho php
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 56

xt nghim v gi tn tt c cc vi khun nu trn trong vng 24 gi (tr Clostridium
botulinum, cn thi gian nui cy tng sinh di). Chng c thit k cho 50 phn ng
PCR vi 50 ng phn ng, cha y thnh phn dung dch m PCR, mi, nc...
gip kim tra chnh xc mc nhim vi sinh vt trm trng hay khng. So vi phng
php nui cy truyn thng, phng php ny cho hiu qu chnh xc v hay hn ch,
n pht hin nhiu vi khun gy bnh hn v tn rt t thi gian.
u im ca phng php PCR:
Thi gian cho kt qu nhanh.
C th nhn din nhng vi sinh vt kh nui cy. vic nui cy tng sinh
l n gin hn v c khi khng cn thit.
Ha cht cn cho phn ng PCR sn c hn v d tn tr hn so vi
trng hp huyt thanh hc. Khng cn dng c v mi trng chn on phc tp, c
th thc hin hin trng.
t tn km v mt nhn s. C th c t ng ha lm gim chi ph
pht hin cc vi sinh vt gy bnh trong thc phm.
Mc d vy phng php vn cn mt s khuyt im cn khc phc:
S c ch hot tnh ca Tag DNA polymerase bi thnh phn ca mu
vt. mu thc phm thng c nhng thnh phn phc tp. vic chit tch v tinh ch
DNA t thc phm hai mi trng trc khi thc hin phng php PCR cho php loi
b nhng hp cht c ch. Tuy nhn mt vi quy trnh pht hin vi sinh vt gy bnh
thc phm bng PCR khng cn tch chit, tinh ch DNA.
Mt vi sinh vt gy bnh hin din trong mu thc phm thng thp,
n trong a s trng hp cn c bc nui cy lm giu c c mt pht
hin bng PCR.
Phng php ny khng phn bit c t bo sng hay t bo cht. do
vy c th dn n trng hp dng tnh gi do DNA t t bo cht. Ngc li phng
php ny cho php pht hin bo t, dng tim sinh, hay t bo cht ca vi sinh vt
gy bnh hoc ng c.
2.3.2.3. Phng php lai phn t
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 57

Hin nay nhiu h thng c thit lp da trn DNA nh lng vi sinh
vt v c t. Tuy nhin, ch c phng php lai phn t (hay cn gi l phng php
mu d, probes) v phng php PCR l c thng mi ha di dng cc b kit pht
hin vi sinh vt gy bnh trong thc phm. Phng php s dng mu d pht hin vi
sinh vt trong thc phm c da trn s pht hin mt on gen c trng ca vi sinh
vt. C s ca vic s dng mu d l phng php lai phn t. Qu trnh ny bao gm
s tch ri hai mch i ca chui xon kp DNA khi nhit vt qu nhit nng
chy (T
m
) ca phn t DNA v s ti bt cp cc trnh t nucleotide b sung khi nhit
tr li bnh thng. S lai phn t xy ra khi on mi c trnh t nucleotide b sung
vi mt vng trnh t trn DNA mc tiu gp nhau do chuyn ng nhit v khi nhit
mi trng thp hn T
m
t nht vi . Qu trnh lai phn t chiu nh hng bi rt
nhiu yu t: nng DNA trong mi trng, nhit v thi gian phn ng, kch
thc cc trnh t lai v lc ion ca mi trng.
V d: h thng d Gen trak (Framingham, USA), h thng ny s dng que
th vi mu d pht hin Listeria trong mu b sa v mu mi trng. mu d l
nhng on oligomer DNA nh du bng ha cht pht quang. Quy trnh phn tch c
th chia lm 6 bc:
1. Ph v t bo thu nhn rRNA
2. Mu d pht hin cha fluorescein isothiocyanate u 5` v 3` ca phn t
c t vo phn ng.
3. Que th c bao bc bi polydeoxythymidine (dT) gn c vi oligodA
ca mu d.
4. Que th c t trong ng o cha mu d pht hin c nh du bng
enzyme
5. Sau khi ra loi phn enzyme tha, que th c t vo ng o cha c cht
to mu.
6. Sau khi hin mu, mu c pht hin bc sng 450nm.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 58









CHNG III
KT LUN V KIN NGH
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 59

3.1. Kt lun
Mc d bnh do Listeria monocytogenes gy ra l tn s thp, 2-5 trng hp
trn mt triu ngi mt nm, nhng t l cht do vi khun ny l rt cao, 25-30%
trng hp t vong trong cc ca nhim bnh.
Phng php truyn thng: Thng quy k thut nh lng listeria
monocytogenes trong thc phm.
-u im :phng php n gin d thc hin.
-Khuyt im: tn nhiu thi gian (vi ba ngy n vi ba tun). chnh xc khng
cao do ngi thc hin c th c sai xt trong qu trnh th nghim.
Png php hin i:
K thut PCR (Polymerase Chain Reaction) vi cp mi LM-F LM-R:
u im :c th pht hin nhanh thc phm nhim trc khn Listeria
monocytogenes vi nng rt thp ch khong 10
2
CFU/g i vi ngi trng thnh
v 10 CFU/g i vi tr em v ph n mang thai.
Khuyt im: Ga thnh kinh t dng thc hin cao v hu ht cc thit
b s dng trong th nghim u phi nhp t nc ngoi.Bn cnh th nghim cn
i nhiu k thut phc tp nn kh nng ng dng khng trong thc t khng cao.
B kit PCR:
u im:
+ Cho php xt nghim v gi tn hu ht tt c cc vi khun trong vng 24 gi.
+ Qa trnh thc hin nhanh gn,cho chnh xc cao.
Khuyt im: Khng s dng c vi Clostridium botulinum v vi khun
ny cn thi gian nui cy tng sinh di.
K thut ELISA : y l phng php hin i cho php xc nh
Listeria monocytogenes mt cch chnh xc v nhanh chng .



Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 60

3.2. Kin ngh
Cc phng php xc nh L. monocytogenes trong thc phm vn cn
nhiu hn ch v mt thi gian cng nh chnh xc khi khng nh s c mt
ca loi ny trong thc phm. v vy nn s dng cc phng php xc nh hin
i nh PCR, ELISA trong phn lp v xc nh vi khun ny nhm m bo
chnh xc cao.
ti ny ch kho st trn mt s sn phm thy sn. v vy ngh tip
tc tin hnh trn nhiu sn phm khc c th p dng phng php ny rng
hn.
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 61

TI LIU THAM KHO
[1]. PGS.TS Nguyn Phng Tin, GS.TS Bi Minh c, GS.TS Nguyn Vn Dp _ Vi
sinh vt trong thc phm k thut kim tra v ch tiu nh gi cht lng an ton
thc phm.
[2]. Trn Linh Thc _ Phng php phn tch vi sinh vt trong nc, thc phm v m
phm.
[3]. H Hunh Thy Dng, 1998 _ Sinh hc phn t, NXB Gio dc.
[4]. V Th Vit Hoa, T Minh Chu, V Th Lm An, Lm Thanh Hin, Nguyn Th
Ngc Dip, Nguyn Thy Hng, 2000 _ Vi sinh vt hc i cng, t sch trng H
Nng lm Thnh Ph H Ch Minh.
[5].http://www.biosyth.com/index.asp?topic_id=178&g=19&m=256
[6].http://www.cdc.gov/epo/dphsi/annsum/1998/98graphs/9844.htm
[7].http://www.en.wikipedia.org/wiki/Listeria
[8].http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnal
yticalManualBAM/UCM071400


Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 62


PH LC
Mt s mi trng chnh trong phng php truyn thng nui cy v phn lp L.
monocytogenes
Thch OXFORD
Code: CM856.
Columbia blood agar base 39.0
Aesculin 1.0.
Ferric ammonium citrate 0.5
Lithium chloride 15.0
pH:7.0 0.2
Cn 27.75g thch Oxford trong 500ml nc ct. Ho tan thch trong nc nng.
Hp t 121
0
trong15 pht.
Lm ngui xung cn 50
0
C v cho thm 1 ng Listeria Selective Supplement
SR:140 ho vi 5ml cn/nc ct v trng(1:1). Trn u, a.
Thch mu cu
Tryptone 14.0
Peptone Neutralised 4.5
Yeast extract 4.5
Sodium chloride 5.0
Agar 12.5
pH:7.3 0.2
Cn 40g thch mu cu trong 1 lt nc ct. Ho tan thch trong nc nng. Hp
t 121
0
C trong 5 pht.
Lm ngui xung 50
0
C v cng thm 7% mu cu v trng.
Thch TRYPTONE SOYA (vi 0.6% yeast extract)
Tryptone 15.0
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 63

Soya Peptone 5.0
Sodium chloride 5.0
Yeast extract 6.0
Agar 15.0
pH:7.3 0.2
Cn 40g thch Tryptone Soya trong 1 lt nc ct. Ho tan thch trong nc
nng. Hp t 121
0
C trong 5 pht.
Mi trng SIM:
Tryptone 20.0
Peptone 6.1
Ferrouss ammonium sulphate 0.2
Sodium thiosulphate 0.2
Agar 3.5
Cn 30g mi trng Sim trong 1 lt nc ct, un tan, ng ng, hp t 121
0
C
trong15 pht. 5. Canh thang ng
Peptone 10.0
NaCl 5.0
Nc ct 1 lt
2% Alconolic B.C.P Solotion 2 ml
ng 50.0
(Sucrose/manitol. . .)
Ho tan cc cht chnh pH:7.2-7.5. San ra cc ng 16 x125mm mi ng 9ml c
cha ng Durhams. Hp t 121
0
C trong 15 pht.
Ghi ch: cc loi ng trn dng phng php lc v trng, khng hp.
Mi trng Clark -Lubs
Peptone 5.0
Glucose 5.0
m
Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 64

Phosphate buffer 5.0
pH: 7.5 0.2
Cn 15g mi trng Clark -Lubs trong 1 lt nc ct. Hp t 121
0
C trong15
pht.
Canh thang TRYPTONE SOYA
Tryptone 15.0
Soya peptone 5.0
Sodium chloride 5.0
Yeast extract 6.0
pH:7.3 0.2
Cn 3g mi trng canh thang Trytone Soya trong 1 lt nc ct. Hp t 121
0
C
trong15 pht.
Nc Pepton
Pepton 1.0g
Nc ct 1000ml v shy; pH=7.2
Cn 1g pepton trong 1 lt nc ct. Hp t 121
0
C trong15 pht.
Listeria selective supplement (Oxford)
(B sung vo mi trng thch Oxford )
Cycloheximide 200 mg
Colistin sulfate 10 mg
Acriflavine 2.5 mg
Cefotetan 1.0 mg
Fosfomycin 5.0 mg
Ly 5 ml nc ct v trng v cn (pha theo t l 1:1), cho vo l lc u, sau ln
vo bnh cu cha 500 ml thch Oxford hp tit trng ngui 50
0
C. Trn u v
vo a v trng.