Cc phng php phn lp v xc nh Listeria spp.

v Listeria monocytogenes: mt cch tng qut

Tm tt:
Listeria monocytogenes l 1 tc nhn gy bnh quan trng trong
thc phm, v c th nghim rng ri cho trong thc phm,
mi trng v cc mu bnh phm.
Cc phng php truyn thng c lin quan c xc nh da
trn s tng trng chn lc v cc c tnh ca Listeria spp
c cy trn a vi sinh vt, da trn hnh thi khun lc, qu
trnh ln men ng v kh nng tan huyt. Nhng phng php
ny l phng php tt, nhng Qu di dng v c th khng
ph hp th nghim cc loi thc phm vi hn s dng
ngn. 1 phng php th nghim nhanh hn c pht trin
da vo cc khng th (ELISA) hoc k thut phn t (PCR
hoc DNA).Trong khi cc th nghim ny c nhy tng
ng, h Th nghim nhanh v cho php hon tt trong vng
48 h. Gn y, cc phng php phn t c pht trin nhm
mc tiu l RNA hn DNA, nh RT-PCR, thi gian thc PCR
hoc axit nucleic da chui khuch i (NASBA). Nhng th
nghim ny khng ch cung cp mt gii php cho kh nng
sng ca t bo, m chng cng c th c s dng phn
tch nh lng. Ngoi ra, mt lot cc bi th nghim c sn,
phc v cho loi c tnh, m l c bit hu ch trong iu tra
dch t.

Phng php u tin phn lp da vo kiu hnh, chng hn

nh chng hn nh multilocus enzyme in di, th thc khun
typing v serotyping.
Nhng phng php kiu hnh ny ang c thay th cho cc
xt nghim phn t, phn nh mi quan h di truyn gia cc
chng phn lp v chnh xc hn. Nhng phng php mi
ny hin nay c s dng ch yu trong nghin cu, n c tim
nng ng k v c kim chng trong tng lai.
I.

Gii thiu

Cc chi Listeria c t trong cc nhnh ph Clostridium

ca vi khun gram dng da trn thp
G + C ni dung ca b gen ca n. C su loi
hin ang c cng nhn:
Listeria monocytogenes,
Listeriainnocua,
Listeria ivanovii,
Listeria seeligeri,
Listeriawelshimeri
v Listeria grayi.

Ch c hai loi chi thng c coi l gy bnh:

L. monocytogenes ngi v L. ivanovii trong khc ng vt
c v.
Tuy nhin, c mt s bo co caL. seeligeri v L. ivanovii
cho thy chng gy bnh con ngi, gy bnh nhim trng
trong listeriosis bi L. monocytogenes v thng nh hng n
con ngi thng qua mt cn bnh tim n nh hng n
h thng min dch, chng hn nh ung th hay AIDS, v d b
nh hng nh ngi cao tui, ph n ang mang thai
, tr s sinh hoc thai nhi. cc triu chng ca bnh ging nh
cm, nhng c th dn n bin chng nghim trng,
chng hn nh vim mng no, nhim khun huyt, h thai hoc
bnh listeriosis ca tr s sinh.
s ca nhim bnh listeriosis trung bnh 40-44 mi nm ti c v
khong 100 mi nm t nm 1993 n nm 1997 ti Hoa K
.Mc d t l mc cc bnh listeriosis
c v nh so vi bnh d ng khc,nhng t vong lin quan l
cao, khong30%.
Mc d Murray ln u tin nghi ng vic ly nhim vi khun
ng vt Nm 1924 l qua ng n ung, nhng phi n nm
1981 ln u tin mt dch bnh bng n c pht hin trong 1
thc phm bi nhim bnh listeriosis. K t cng nhn

L. monocytogenes l mt tc nhn gy bnh, truyn qua thc

phm, y l bc tin quan trng trong s pht trin ca
phng php phn lp v xc nh listeria. n lc ban u
c lp Listeria t thc phm da trn cc th tc lm sng
nh cy trc tip ln mi trng thch mu l khng thnh
cng.
pht trin ng k nht khng ch xy ra trong th tc chn lc
phng php m cn c s sn sng ca nhiu ngi mi v
nhanh chng pht hin ra phng php da trn khng th v
cng ngh phn t.
mt thch thc ln cho thc phm c kim nghim l c s
h tr ca cc th nghim bng 1 phng php mi l c ch
cc thnh phn trong thc phm, chng hn nh kim st s
min dch, c pht trin lm sch cc cht t cc thnh
phn thc phm c ch cng nh tng tnh linh hot.
Hin nay c mt phng php tm kim bit thm nhanh
chng v Linh hot, c bit l trong ngnh cng nghip thc
phm, l ni cung cp cht gy nhim thc phm min ph
v tung ra sn phm d h hng ra th trng trc ngy ht hn
ca chng, nui cy v th nghim l cc phng php c s
dng rng ri bi v n n gin, r tin v cho php to ra 1
lng mu ln.
Xt nghim cc khng th da trn L. monocytogenes-

Protein, s dng phng php nui cy c th kim tra

bng cht o mu hoc cht pht quang trong mi trng
pht hin c t ca loi gy bnh v khng gy bnh.
Mt php th rng ri nht c s dng trong cc th nghim
da trn mt s tnh nng quan trng c cung cp trong bng
8 v mt ci nhn khch quan ton din, nhn dng
v phng php nh du cho Listeria v L. monocytogenes
trong thc phm v cc mu mi trng c cung cp trong
hnh. 1.
Nhiu cuc th nghieemk xc nhn v quy nh ca chnh
ph cc c quan nh Cc Qun l Thc phm v Dc
(FDA) ,S Nng nghip M (USDA) Hoa K ti
Hoa K, c v New Zealand, Qun l thc phm (ANZFA) ti
c, khng phn bit c gia cc loi Listeria.
T gc nhn v sinh thc phm th c s hin din ca mt loi
khng gy bnh nh L. innocua c th ch ra nguy c nhim
vi L. monocytogenes. Tuy nhin, Cc nghin cu dch t hc
cho thy rng ch c L. monocytogenes v ch chng thuc
serotype 1 / 2a, 1 / 2b v 4b tham gia vo 90% ca s bng
pht ca bnh listeriosis.
****************************************************
**************

v tn huyt do L. ivanovii c tng cng trong vng ln cn

ca R.equi vt. Tuy nhin, cc th nghim CAMP c cu ng
dng ca n v n ch ra rng i khi n khng th phn bit
chnh xc gia L. monocytogenes v L. ivanovii [17]. Do ,
mt bin th ca th nghim CAMP gc bng a thng mi
c khuyn co trong cc phng php USDA [11,14]. Vic
xc nh cc loi khng tn huyt da trn s khc bit trong
qu trnh ln men ng nht nh (Hnh 2). Trong khi cc im
nh du nhn dng sinh ha l hu ch, chng l i khi rt kh
gii thch v cc phn ng mu sc c th m h.

3.3 Cht nn to sc t
Mt s pht trin gn y l sn xut v thng mi sn c ca
phng tin truyn thng to sc t. Xc nh nhanh cc enzyme
ca vi khun c cung cp bi vic s dng cc cht nn to
sc t, c a vo phng tin truyn thng m cho php
nhn dng trc tip ca cc khun lc bi mu sc c trng ca
chng. Phosphatidylinositol c th phospholipase C (PIPL-C) l
mt enzyme ch c sn xut bi L. monocytogenes and
L.ivanovii [18,19] v hot ng ca enzyme ny c o bi
phng tin truyn thng to sc t. Nhiu phng tin truyn
thng to sc t l thng mi sn c v ang dn dn c
chp nhn bi c quan qun l [11]. y l nhng phng tin
truyn thng cung cp nhiu li th hn cc xt nghim khc.
Chng n gin, hiu qu chi ph, d dng gii thch, cho
php mt lng mu ln, rt nhy cm v c th, v c th c

thc hin trong khong thi gian tng t nh phng php

ELISA (Bng 8). Mt thch to sc t thng mi c sn pht
hin L. monocytogenes l thch Rapid'L.mono (BioRad,
Marnes de la Coquette, Php) trn c vi khun PIPL-C dng
to khun lc mu xanh. Cch phn bit L.monocytogenes t L.
ivanovii c thc hin bng qu trnh ln men ca xylose
(Hnh 2). Cc th nghim ca Rapid L.mono c nh gi
rng ri trn mt lot cc mu khc nhau [20,21]. Kim tra thch
thng mi c sn khc da trn s pht hin vi khun PIPL-C
tch cc l nhng th nghim BCM thch to sc t (Biosynth
quc t, Naperville, M), th nghim CHRO-Magar Listeria
(Mast Diagnostics, Reinfeld, c), v ALOA (Biolife, Milan,
). Tuy nhin, khng ai trong s nhng th nghim phn bit
gia L. monocytogenes v L. ivanovii [22-26]. Mt enzyme vi
khun c s dng cho vic xc nh cc L. monocytogenes l
alanyl peptidase, c sn xut bi tt c cc loi Listeria ngoi
tr L. monocytogenes. Mt phn ng mu sc n gin c s
dng trong cc cht nn DL-alanine--naphthylamide v Dalanine-p-nitrianilide b thy phn [27], v l c s cho cc ID
Disc Monocytogenes (Biolife, Milan, ).

3.4 Xt nghim khng th da trn

Phng php xt nghim min dch da trn khng th c hiu
vi vi khun Listeria c p dng trong kim nghim thc
phm trong nhiu nm v chng c ph bin bi v xt
nghim c th c thc hin trc tip t phng tin truyn

thng lm giu m khng chun b mu t nht. Nhiu trong s

cc xt nghim min dch c sn nh b dng c thng mi v
c s chp thun ca c quan qun l [15], Bng 1 v 8.
3.4.1 Enzyme lin kt kho nghim min dch (ELISA)
Phng php ELISA s dng mt khng th c nh vo mt
ging chp khng nguyn kt hp vi mt khng th th cp
cng mt loi enzyme (hoc nhn khc) pht hin khng
nguyn bt gi, l nhng phng php p dng rng ri nht bi
v h kt hp d s dng vi cc th h Kt qu xt nghim
nhanh. Hn na, phng php ELISA c th c s dng vi
cc ma trn mu kh m lm cc xt nghim ny c bit thch
hp cho kim nghim thc phm. Mt pht trin gn y l s
sn c ca ngy hm sau kim tra Listeria cho cc phng
php thc phm v vn ha mi trng v cho php mt kt qu
trong vng 30 gi k t khi nhn mu (Listeria c o, TECRA
quc t, Frenchs rng, c v Vidas Listeria Express,
bioMerieux, Marcy Etoile, Php) (Bng 1 v 8).
3.4.2 Chp min dch
Chp min dch l mt k thut thanh lch m s dng ht t
tnh (Dynal, Melbourne, c) hoc que nhng (Listeria c o,
TECRA quc t, Frenchs rng, c) c ph khng th c hiu
tch Listeria t vi cnh tranh v componets thc phm c
ch. Phng php chp c th nh th ny cng tp trung cc
sinh vt mc tiu v tng nhy ca cc th nghim [28]. K
thut chp c th c lin kt vi cc phng php phn t

tng sc mnh ca phn bit i x vi mt mc phn loi

[29-32].
S khc bit ca L. monocytogenes t loi Listeria khc da trn
khng th c hiu vi c lc yu t c th hin bi L.
monocytogenes gt hi thnh cng bin. Trong khi c
nhiu ti chng li cc yu t c lc [33], nhng khng c,
trong hu ht cc trng hp, c hiu qu th nghim. iu
ny ch yu l do biu hin yu t c lc trong ng nghim l
cc k bin [34-40]. Tuy nhin, mt ngoi l l kho nghim
VIDASLMO (bio-Merieux, Marcy-Etoile, Php), m mc tiu
thnh cng mt khng nguyn c lc n nh lm c s cho L.
monocytogenes c xt nghim hunh quang lin kt enzyme
(EFLA).

3.5 Xt nghim phn t

Xc nh Listeria spp. v L. monocytogenes s dng phng
php phn t ngy cng tr nn ph bin v nhng k thut ny
l cc k chnh xc, nhy cm v c th. Xc nh v phn bit
ca L. monocytogenes t loi Listeria khc n mt cp phn
loi c th c thc hin trong khong thi gian tng t nh
xt nghim ELISA da trn.
3.5.1 Lai AND
Lai DNA l phng php phn t n gin c s dng
pht hin Listeria v L. monocytogenes trong thc phm (Bng

2). S hin din ca mt chui mc tiu c pht hin s dng

mt u d oligonucleotide ca chui b sung cho trnh t DNA
mc tiu c s dng trc nh cc nhn pht hin.
Gn y hn, u d sinh hc tinylated, thm d kt hp
digoxygenin (Boehringger-Mannheim), hoc nh du hunh
quang cho php pht hin cc trnh t mc tiu vi nhy
tng ng vi u d phng x, nu khng c s mm bnh
lin quan ti phng x. PCR kt hp vi ADN trong mt phin
vi l mt cch tip cn thun tin v rt nhy cm v c th
pht hin Listeria trong mt thng lng cao nh dng 96-tt
[57]. Xt nghim ADN nhm mc ch ch yu phn bit cc
L. monocytogenes t loi Listeria khc bng cch tn cng tu
thm d c gen yu t (Bng 2). Thng mi c sn cc xt
nghim ADN thng c s dng cho s kim nh thc phm
v c th nghim rng ri cho nhy v chnh xc ca
chng [58]. Cc xt nghim ny mnh m v ng tin cy.
Accuprobe (Gen-Probe Inc., San Diego, Hoa K) l mt th
nghim da trn lai ca thit b thm d DNA dn nhn c
t mRNA, do m bo rng ch c t bo sng c pht
hin [59-61]. Mt th nghim thng mi l lai kit GeneTrak
DNA (Neogen Corporation, Lansing MI). Mt th nghim
thng mi gn y tr nn sn c s dng cng ngh nhn
dng vermicon (VIT, Munich, c) v c da trn lai to
ti ch ca cc u d oligonucleotide hunh quang dn nhn
RNA mc tiu bn trong t bo. Xt nghim ny c s
dng cho n nay ch duy nht cho vic xc nh cc L.
monocytogenes trong cc mu nc thi v bn [62].

****************************************************
************
3.5.2 Phn ng chui Polymerase (PCR)
PCR c mt nh hng ln trn tt c cc ng dng
phn t k t khi c gii thiu. PCR l mt k thut trong
cc phn on ca DNA c khuch i bng cch s
dng nhit n nh DNA Polymerase v hai on mi (DNA
ngn trnh t c th gen c bit) v mnh v c khuch
i sau c pht hin thng s dng agarose gel in.
Tri ngc vi DNA, ni mt lng tng i ln cc DNA
mc tiu hoc RNA c yu cu thc hin cc kim tra.
PCR khuch i mt lng ln DNA t mt lng nh DNA
mc tiu. PCR hin nay c thnh lp nh mt k thut
ng tin cy v ti sn xut xc nh Listeria spp v quan
trng hn phn bit cc ocytogenes L.mon t loi listeria
khc s dng mi nhm mc tiu ca cc yu t c lc or
RNA ph cho n gen (bng 3). Pht hin bng PCR c
thc hin mu sau khi chn loc c c (lm giu)cho 24-48h.
N c chng minh rng xt nghim trc tip ca mu
m khng lm c c trc khi cho kt qu khng ng tin
cy [77,89,90]. Mt tr ngi ln cho vic s dng k thut
PCR trong cc mu thc phm v mi trng trc tip sau
khi tng sinh chn lc l nc xut v cc thnh phn thc

phm c chc cht c ch PCR dn n kt qu m

tnh gi tip cn [91]. Sev-eral c th c thc hin loi
b c ch fac-tor sau khi lm phong ph la chn nh x l
mu [92-96] hoc th tc cch ly s dng ht mag-di truyn
que nhng hoc mng nhm DNA t cc ma trn phn ng
c ch [29-31,62,99-102].
a hp PCR ni nhiu cp mi c s dng cho php pht
hin ng thi nhiu hn mt tc nhn gy bnh tronh mu
th nh Listeria v Salmonella [29,31] hoc
L.monocytogenes v loi Listeria khc [101,103-106].
Phng php ny l hp dn nht phn tch thc phm
m thi gian th nghim ha cht v chi ph lao ng s
dng nhiu cp mi c lng PCR. Tuy vy cc cp mi
c s dng trong phn ng lin tc v c thit k vi
cng mt mc tiu. N c s dng o nhy cm v
c hiu trong nhn v c s dng cho vic xc
nh cc L.monocytogenes trong cc mu lm sng [57,107110], mu mi trng [78] v cc mu sa [97,111,112]. Xt
nghim PCR thng mi sn c h thng BAX-PCR
(qualicon, wilmington,DE,USA) v kho nghim Probelia
(sanofi chun on Pas-teur, Marne la Coquette, France)
c tri-aled trong lnh vc ny trn nhiu loi khc nhau
loi mu [113-122].
4. Phng php nhn dng c s dng trong dch t hc
Pht hin Listeria trong cc mu thc phm, mu mi
trng v bnh x ical s dng phng php sinh ha v

huyt thanh lc, cng nh mt s phng php phn t

thng xc nh Listeria gy nhim n mc loi. Ch
L.monocytogenes c coi l mt tc nhn gy bnh ca con
ngi v ch c 3 type huyt thanh ca L.monocytogenes c
lin quan n dch bnh Listeria truyn qua thc phm ln.
Do iu tra dch t phi s dng cc k thut c kh nng
phn bit chng L.monocytogenes lin quan cht ch c
th xc nhn ngun dch, thit lp m hnh truyn ti , xc
nh v theo di cc h cha cc chng i dch. iu tra
dch t hc c da trn gi nh rng cc chng vi sinh
vt thuc iso-lated t cc ngun khc nhau l dng v tnh
(h) lin quan v tng t hoc ging ht nhau trong kiu
hnh v c im molecu-lar. Do cc phng php ny
c da trn cc protein c hiu loi hoc gen tng i
n nh theo thi gian v c truyn t th h ny sang th
h khc. Ngoi h thng nh my phi c kh nng phn
loi chnh xc tt c cc chng dch t li lated mt dch
theo dng v tnh rela-mi (dch t khp) [164]. Ngoi nhng
yu cu v nhy v c hiu ca xt nghim c nhng
cn nhc khc a vo ac-count khi la chn mt th
nghim thch hp s dng trong epi-demiology, chng
hn nh s d dng trong s dng cc phng php v gii
thch kt qu lao ng v vt liu chi ph sam kh nng
thng ple thi gian thc hin thc hin cc bi kim tra
cng nh s n nh v tnh lp li ca cc th nghim bn
thn

4.1.phng php nh my kiu hnh

nh my ca chng L.monocytogenes hoc l da vo
du kiu hnh, chng hn nh soma O-v flagella H-yu t
g huyt thanh hc, th th thc khun (phage nh
my) hoc cc enzyme (enzyme multilocus in), hoc h
c da trn cc bin phn t trong b gen ca vi khun
(phng php nh my phn t).
4.1.1. G huyt thanh hc
g huyt thanh hc c da trn khng th tit hocically phn ng vi soma hoc "O" khng nguyn v flagella
hoc "H" khng nguyn (bng 4) ca cc loi Listeria.
Listeria spp c nhm li thnh cc tp huyt thanh theo
O hoc H khng nguyn c hin th. Tp huyt thanh
c chia s gia cc loi Listeria v cc khng th chng li
cc khng nguyn ditected cho php xc nh cc chi
Listeria nhng khng phn bit Listeria species.Athough,
serologi- cal xc nhn l khng cn thit cho vic xc nh
cc L.monocytogenes, ngay c i vi cc mc ch quy
nh, serol-ogy l thng c s dng xc nh s ph
bin ca cc chng loi trong cc nghin cu dch t hc v
theo di nhim mi trng [165.166]. Mt kit thng mi
cho serotyping Listeria phn lp c sn t denka Seiken
(tokyo, japan) v khng huyt thanh cng c th c ly t
difco (Difco laborotories / Becton Dick-inson v Co, Sparks,
MD, USA).

4.1.2 Phage buc

phage nh my l mt phng php buc cng nh
thnh lp c mnh m nh gi trn mt lot cc mu
(bng 5). N c c bit hu ch trong vic thit lp
thc phm l chic xe chnh cho nhim Listeria [167-170].
Phng php ny da trn cc loi Interac-tion ca mt vi
khun c bit vi chng Listeria ch ca n, dn n ly gii
t bo ch. Nm 1985, vic s dng mt tp hp cc th thc
khun c xc nh (quc t) vi cc chng ch ca mnh
tiu chun ha thc khun g c xut bi
Rocourt v ng nghip [171]. Mt bt li ln ca k thut
phage nh my l khng phi tt c cc chng
L.monocytogenes l typ c. c bit L.monocytogenes
serotype 1/2 chng c mt typa-trch thp so vi cc tp
huyt thanh khc [172.173]. K t L.monocytogenes 1/2
chng l thc phm ph bin contam-inants v mt trong
nhng chng chu trch nhim v s bng pht bnh
listeriosis, y l mt hn ch nghim trng trong vic s
dng thc khun-typ-ing ca L.monocytogenes phn lp cho
cc nghin cu epiemiological. N cng c ghi nhn
rng typability ca chng bng cch s dng b quc t
khc vi cc a im geogpraph-ical cc chng v iu ny
c th ng gp cho nhng khim khuyt ca h thng nh
my [173.174].
4.1.3 multilocus enzyme in di

MEE cng l mt k thut nh my cng c thnh lp

(bng 6). N c da trn vic quan st s bin i trong
trnh t axit amin ca cc enzym dn n mt in tch tnh
in khc nhau ca protein, m trong kt qu ln lt trong
mt di chuyn in di khc nhau. nhng bin i trong tnh
di ng c lin quan trc tip n alen bin th ca gen m
ha cho cc enzyme ny. Cn c vo tnh di ng in
phoretic ca h, phn lp c th c sp xp thnh cc loi
electropho-retic. th tc n gin v lin quan n vic
chun b ca cc chit xut enzyme do ly gii t bo vi
khun, in di cc cht chit xut s dng gel tinh bt v
nhum c th ca cc enzym
phage nh my v MEE ni chung c chng minh l
phng php ng tin cy v thun tin, trong th hin
tt repro-ducibility khi c s dng trong nhiu nghin
cu lin phng th nghim ca WHO [176]. nhng phng
php ny l d s dng.

****************************************************
*************************
4.2
k thut nh my phn t da trn ADN lai, PCR, phn tch
enzyme hn ch hoc gii trnh t trc tip ca ADN. Trnh t
trc tip ca ADN l cch chnh xc nht so snh s khc bit
di truyn hoc tng ng. Tuy nhin, n cng l t tin v
thi gian nht phng php tiu th, v hin ti khng th c
p dng kim tra lng mu cao. Do , phng php cho
mt s phn nh tng i chnh xc cc bin th di truyn cng
nh mt lng mu cao trong mt khong thi gian nhanh
chng c pht trin. Nhng phng php ny nhm thit lp
cc mc bin th alen ca cc gen c bit, sau hnh thnh
c s o lng Quan h di truyn ca cc chng vi khun
Listeria. bin th alen c th c o nh cc bin th trong
chiu di ca mnh ADN c th c to ra, hoc bng cch tiu

ha hn ch hoc PCR, hoc nh l mt s thay i trong cu do

s khc bit trnh t (v hnh dng a hnh)
din gii d liu vi chnh xc cao hn, k thut in
di c pht trin cho php phn gii tt hn cc mnh
ADN, chng hn nh lnh vc xung in di gel (PFGE), m ch
yu c s dng kt hp vi hn ch enzyme (endonuclease)
tiu ha ca ADN. Bin tnh in Gradient gel (DGGE) hoc
in di mao qun (CE) l k thut in di c s dng kt hp
vi cc n si cu hnh khng a hnh (SSCP) phn tch
pht hin bin th nucleotide. Nhiu ngi trong s nhng k
thut nh my phn t tip tc c vn ngn chn s dng
thng xuyn. y ch yu l do thiu s chun ha cc thng
s th nghim c nhn m c th dn g chnh xc v ngho
tng quan lin phng th nghim ca cc kt qu. thit b Hn
na, mt s trong nhng bi kim tra yu cu chuyn ngnh, ti
liu v cc thuc th v th nghim l tn km khi thc hin
trn mt quy m ln l cn thit cho hu ht cc iu tra dch
t hc
Cc phng php phn t quan trng nht c s dng
g ca ca L.monocytogenes c m t trong bi nh gi ny.
Mt s k thut nh ribotyping, tiu ha macrorestriction,
PFGE, PCR-K thut RFLP (RFLP) hoc ngu nhin khuch i
DNA a hnh (RAPD), c s dng thng xuyn cng nh
thnh lp. Cc k thut khc nh SSCP hoc g chui
multilocus (MLST) hin ang tr nn thnh lp nh l k thut
nh my cho L.monocytogenes v cho thy nhiu ha hn. Tuy

nhin, h khng c s dng thng xuyn v khng c nhiu

d liu lnh vc tn ti cho nhng phng php
4.2.1
Ribotyping c da trn cc bin th gen ribosome hoc
protein. Phng php ny c s dng thit lp cc mi
quan h pht sinh loi v l c s cho h thng hc hin i ca
prokaryote. Mi quan h ca sinh vt c nh gi tt nht bi
cch cht ch trnh t ADN trong mt trn u gen nht nh.
Cc gen hu ch nht nh gi cc mi quan h pht sinh loi
l gen m ha cho ARN ribosome v gen ribosome c mt trong
tt c cc sinh vt trong nhiu bn trn ton b gen v ribosome
chc nng c coi l khng i theo thi gian biu tin ha
lu di. Ni chung, ribotyping Listeria chng lin quan n vic
hn ch enzyme tiu ha ca nhim sc th ADN sau ADN lai
s dng mt u d gen rARN. Kt qu m hnh di c s
dng sp xp Listeria phn lp thnh ribotypes v thit lp
mi lin h ca cc phn lp. Rybotyping c s dng rng
ri trong cc nghin cu dch t hc v t ng ha cho php
s thch ng ca phng php ny phn tch thi quen. Mc
d phng php ny l thun tin, mnh m v mang li kh
nng ti xut sc sc mnh ca phn bit i x i vi
L.monocytogenes g c bo co l thp hn so vi cc k
thut nh my phn t khc

4.2.2

k thut ny lin quan n vic s dng cc enzym hn ch

nhn ra v ct trnh t c th trong phn t ADN. Cc tiu ha
ca ADN to ra mt m hnh di cc mnh vi kch thc khc
nhau. Quan h di truyn c xc nh bng cch so snh s
lng v kch thc ca cc mnh v c tch ra v hnh dung
s dng gel in di. REA ca nhim sc th ca vi khun ADN
c gi l phn tch macrorestriction v vic thc hin cc k
thut c tng cng ng k bi s kt hp vi PFGE. Trong
loi in, phn t ADN c chu thay i nh k theo hng
v / hoc cng ca hin ti ca in trng p dng. phn t
ADN c th c tch theo kch thc ca chng v cc phn t
nh hn p ng tt hn vi nhng thay i trong lnh vc in
hn so vi cc phn t ln hn. S dng phn t in di ADN
thng thng ln n 20 kb c th c tch ra, trong khi cc
phn t ADN ln n 12MB c th c tch ra v phn tch bi
PGFE. S kt hp ca macrorestriction tiu ha vi PFGE c
coi l phng php nh my hiu qu nht v n l n gin,
thi gian v chi ph hiu qu, v c phn bit i x. Vi cc
iu chnh ch nh k thut ny c th c p dng cho hu
nh bt k vi khun hay loi chng. Trong cc nghin cu so
snh vi cc phng php nh my khc n c tm thy
l cc th nghim phn bit i x nht, c bit l cho nh
my ca L.monocytogenes serotype 1/2 v 4
4.2.3
hai phng php PCR bsaed khc nhau c th c s dng
loi chng. Mt cch tip cn (RAPD) s dng cc on mi

ngu nhin khuch i cc on ADN ngu nhin. Phng

php th hai l khuch i trnh t mc tiu c th v phn
tch sn phm PCR hoc tiu ha enzyme gii hn ca h, bng
cch so snh di ca ADN mnh (RFLP) hoc cc bin th v
hnh dng (SSCP) trong cc sn phm PCR. Trong k thut
RAPD gen ADN c c trng da vo s lng v kch thc
ca cc mnh ADN khuch i c to ra bng cch s dng
mt mi ngu nhin hay ph qut duy nht trong mt PCR.
Nhng thay i nh trong ADN di truyn s dn n kch thc
v s lng cc mnh v khuch i khc nhau. K thut ny c
nhiu u im so vi PCR thng thng. Cc mi s dng
thng l 8-10 bp di v trnh t ngu nhin, nhng kin thc v
mc tiu ADN t l khng cn thit. Cc mi tng t c th
c s dng cho nhiu loi vi khun khc nhau ch vi nhng
thay i nh trong giao thc. Ni chung t nht ba mi ph c
s dng trong cc phn ng ring bit cho ngn tay-in, v cc
mu di tip theo c so snh cho mi mi. Vic ng dng cc
k thut g cc loi Listeria v ph nh my ca chng
L.monocytogenes c ghi nhn trong nhiu nghin cu t
nhiu phng th nghim khc nhau v cng c th hin
trong mt cuc th nghim lin phng s dng ba mi ph qut.
RAPD nh my l mt cng c tuyt vi cho cc nghin cu
dch t hc v c s dng rng ri lin kt cc chng
phn lp t L.monocytogenes trng hp bnh listeriosis cho
thc phmkh nng p dng cc k thut g cc loi Listeria
v ph nh my ca chng L.monocytogenes c ghi nhn
trong nhiu nghin cu t nhiu phng th nghim khc nhau v

cng c th hin trong mt cuc th nghim lin phng th

nghim bng ba mi ph qut. RADP g nh mt cng c tuyt
vi cho cc nghin cu dch t hc v c s dng rng ri
lin kt cc chng phn lp t L.monocytogenes trng hp
bnh listeriosis cho thc phm m dnh lu vo v dch.
RAPD c s dng rng ri g c phn lp t cc nh
my gia cm, nh khi, mi trng sa, ch bin tht ln v cc
nh my git m v loi tr hoc bao gm cc sn phm thc
phm lin quan. Nhng li th ca RAPD g so vi methids
phn t khc l s n gin ca cc kim tra, tc m vi khun
Listeria c th c g vo mt mc phn loi v kh nng
sng lc s lng mu ln
Ngc li vi RAPD nh my, PCR-RFLP lin quan n s
khuch i PCR ca gen mc tiu c th v tiu ha ca sn
phm PCR vi mt enzyme hn ch. Cc mnh v hn ch c
tch ra bng cch so snh s lng v kch thc ca cc mnh
v. gen mc tiu thng l cc gen m ha cho ribosom tiu n
v hoc cc yu t c lc. Trong khi phng php ny l ph
bin do thc t l n cho php mt lng mu ln trong mt
khung thi gian ngn kt hp vi tnh d s dng v gii thch
cc kt qu, kh nng phn bit ca phng php PCR-RFLP l
khng cao v do vic s dng n trong iu tra dch t hc
c gii hn
phn tch a hnh cu hnh khng c da trn gi nh rng
mt s thay i trong trnh t nucleotide ca mt sn phm PCR
s dn n mt s thay i trong cu to ca mt si duy nht

ca sn phm . thay i v hnh dng ny c th c pht

hin bng gel in di v cc mnh ADN bin tnh ca trnh t
khc nhau s di chuyn tc khc nhau. nghin cu ban u
ca SSCP s dng khng bin tnh gel polyacrylamide phn
tch, nhng gn y bin tnh (DGGE) hoc gradient nhit
gel in di (TGGE) c s dng mt cch hiu qu hn
tch cc mnh ADN m c th khc nhau ch trong mt vi
nucleotide. in c thc hin bng cch gii thiu cc tc
nhn bin tnh, chng hn nh ur v formamid, vo mt gel
polyacrylamide (DGGE) hoc bng cch p dng mt gradient
nhit trn gel (TGGE) m mt phn lm bin tnh gp i b
mc kt ADN. S khc bit v trnh t nucleotide s dn n
hnh vi nng chy khc nhau ca cc on ADN v do ADN
s di chuyn vi mt tc khc nhau thng qua cc gel.
L.monocytogenes chng phn lp t cc loi thc phm c
nh my s dng k thut ny. . Tuy nhin, khi in di gel p
dng ph bin nht trong vic iu tra s dng SSCP g l mao
mch in (CE). SSCP-CE c kh nng phn bit cc mnh
ADN, khc nhau ch bi mt nucleotide. Tch cht phn tch
c thc hin trong cc mao mch silica c lm y vi mt
ma trn sng. ADN mnh v c pht hin di nh sng tia
cc tm khi h vt qua mt my d vo cui ca mao mch.
qu trnh ny l hon ton t ng v vay chnh n sng lc
thng cao. l mt la chn hu ch v hiu qu chi ph trc
tip ADN seuqencing, ch c cc phng php khc c kh nng
pht hin cc nucleotide polymorphisms. L.monocytogenes

c nh my bng SSCP ca 16S rADN v hemolysin (hly)

v gen IAP
****************************************************
*************
5. Tng lai hng cho v pht hin xc nh
L. gien n nhn
Phn sau y xem xt phng php tiu thuyt cho ofListeriaspp pht hin. thc n,
mu lm sng v mi trng. Phng php ny hin c thu bng nghin cu
chuyn dng phng th nghim ch ; nhng, v phng php ny a ra li th ng
k trn k thut thng thng, h c th l s dng thng xuyn cho th nghim
mm bnh thc n trong tng lai.
5.1. Xt nghim nhm n Axit ribonuclenic
Th nghim thc n hoc mu vt mi trng dnh ring cho patho genicListeria
nn nhm n c th sng v ch liveListeriacells mi c th gy ra bnh. Kh nng
to thnh thuc a trn thch rn l vng tiu chun phng php nui cy xc
nhn hin din ca mm bnh sngV y l tiu chun da vo tt c kim tra
pht hin Listeria khc c so snh vi nhau. Xt nghim nhm n DNA nh l
PCR b ch trch v c quan cht - hc thuyt c th cho kt qu kh quan v n
nh ca phn t ADN kh cao. La chn ca Axit ribonuclenic hoc mRNA nh mc
tiu cho th nghim mm bnh thc n c t hp gia tng thin v t hin din ca
mRNA l du hiu ca sng tnh trng t bo [ 272,273 ].
mRNA l khng bn phn t v l lng xung cp sau khi t bo t vong do RNases
v yu t mi trng nh l sc nng. Khc thun li ca th nghim cho mRNA l
nhiu bn sao ca gien mc tiu c mt, ln lt ci tin nhy xt nghim. Bt
n ca cc phn t mRNA, mc d li th, cng c th l s bt li v th nghim
phi thc hin theo iu kin ngn nga suy thoi ca ch. S bt li khc v s
dng Axit ribonuclenic - da vo cng ngh l chi ph cao thit b v cht th, v
nhn vin phi rt c hun luyn thc hin v nh gi xt nghim. nhy cm
v c trng ca xt nghim Axit ribonuclenic c bo co c tng ng
hoc hay hn PCR chun [ 274 ]. Nhiu
Axit ribonuclenic - da vo xt nghim c thc hin trong 96 - ging dng thc
no ph hp cao - thng lng th nghim t ng-thng, yu t lm cng ngh
ny hp dn cho ng dng hng ngy tng lai thc n th nghim v dch t hc
iu tra.
5.1.1. Bn sao o ngc ( RT ) - PCR

RT - PCR bao gm phn ng hai bc, vi bc u tin s dng en - zim

transcriptase li nghip v kinh t pht sinh trn - mRNA tr vo ADN b tr
( cDNA ). Phn ng thng thc hin bi on ADN ngn ngu nhin. bc th hai
cDNA c dng lm tem - a cho s khuch i ca dy c th bng PCR s dng
on ADN ngn c th mc tiu v
En - zim tng hp ADN to iu kin thun li phn ng. L. monocyt - ogeneshas
c pht hin s dng phng php ny trong mu tht ghp bng phng php
nhn to [ 275 ] bng nhm n mRNA tran - script ca thehly, gien prfAandiap,
trong mu cht thi
[ 276 ] bng nhm n bn sao cho gien rRNA, cng nh cho pht hin ca sc
nng - injuredL. gien n nhn bng nhm n the hly transcript [ 277 ].
5.1.2. PCR v ngay tc th
K thut ny l iu chnh RT - PCR phng php. Trn phn ng cha lm hng
qunh quang nh du ( SYBR Green ) rng buc c th tng gp i DNA b
lm vo hon cnh kh khn. Tng pht hunh quang sau khi mi chu k lin tip
cho php nh lng trc tip ca mc tiu
DNA. Loi phn tch ny i hi trang b - ment chuyn dng v vt cht m ng
k tng chi ph th nghim. Phng php ny c s dng nhn bit v nh
lng L. monocytogenesin thc phm v mu lm sng trong vi nghin cu
[ 74,154,278-281 ], v cho pht hin ng thi ca ListeriaandSalmonella
[ 151,152 ]. Thnh lp thc t s lng contami - 864 U. Gasanov v nhng ngi
khc/ Bi ph bnh Vi sinh hc FEMS 29 ( 2005 ) 851-875
nating monocytogenescells L. thc n v bao quanh - tm thn mu l yu t quan
trng khi o ngc kh nng pht bnh ca bnh nhim khun lm nh hng ln
h thn kinh. PCR v ngay tc th l nh lng, l li th ng k hn cc phng
php phn t, cho nn cng ngh ny l v cng hp dn cho th nghim thc n
v iu tra dch t hc.
5.1.3. Dy a - xt nu - cl - ch - da vo s khuch i
( NASBA )
NASBA l thay th cho PCR thng thng v da vo hnh ng ca ba en - zim.
bc u tin transcriptase o ngc c dng trong s kt hp vi oli gonucleotide tc nhn ban u to ra cDNA - Axit ribonuclenic ging lai phn t.
bc tip theo en - zim RNase a Axit ribonuclenic ra khi phn t ging lai
transcriptase o ngc tng hp phn t cDNA b lm vo hon cnh kh khn gp
i. CDNA ny l sau c dng lm mu cho th h bn sao Axit ribonuclenic
bng T7 polymerase.

Tri vi PCR, NASBA mang li mt chui

Moleules axit ribonuclenic m c th pht hin hoc do thng thng agarose in
di hoc bng cch s dng nh du chui p - li - me ngn tu thm d cho xt
nghim lai ging kt hp vi h thng pht hin cng mu. Mc d
NASBA l phng php cho thy tim nng ln cho cng vic hng ngy phn tch
thc n v mi trng th nghim
[ 273,282 ],
c mt s khc bit vi thng thng phng php PCR c bit lin quan n s chun b
mu -tion , m phc tp k thut ny. axit nucleic phi c trch trc khi th nghim, bi v
khng ging nh PCR , phn ng c thc hin ti mt hng s nhit ca 41 C , m
khng cao vi khun ly- sis v th nghim do trc tip ca mu lm giu c khng
kh thi. Mt khc , k t khi cc phn ng l mi hnh thnh nhit khng i xe p nhit
trang thit b l khng cn thit. NASBA c s dng cho cc pht hin ofL . thc phm
monocytogenesin [ 283.284 ] , v c tm thy l ca nhy tng ng , nhng tt
hn c hiu, hn l vn ha truyn thng v ELISA da trn(284)
5.2. microarray DNA
microarray DNA l mt cng ngh mi xut cnh,
m l da trn DNA hoc RNA lai. DNA
microarray c s dng iu tra s pht trin ca vi sinh vt
v dch t hc v c th phc v nh mt cng c chn on
cho lm sng th nghim, mi trng hoc thc phm. H l
v c bn o ngc dot-blots v c sn xut bi ch-ting DNA (thng l 100200 lm kch thc 200-500lm
ngoi) vo mt ma trn h tr vng chc [285]. microarray
l thng mi c sn t nhiu cc nh cung cp khc nhau (Applied Biosystems,
Affymetrix, Qiagen v nhiu
nhng ngi khc) v c th c ty chnh thc hin cho cc mc ch c th.
C hai dng microarray chnh, mt l da trn trn oligonucleotide c th trnh t v
cc sn phm PCR c th cc nhn vin khc.
5.2.1. microarray PCR da trn
to microarray PCR da trn n l cn thit
khuch i vng c th quan tm s dng mc tiu c th

mi. microarray ny thng c xy dng

i din cho ton b h gen ca vi khun v mi l
thit k che cc khung c m cng nh vng lin genic. Sn phm PCR c
tinh ch trc khi gt bt b mt vo h tr, slide knh thng trng hoc
mng, sau c s dng lai to stud-t vi DNA mc tiu. Cc mc tiu ADN
t mu
c to ra t tng s RNA ca vi khun s dng ngu nhin hoc
mi c th v RT-PCR. Nhng sn phm PCR l
sau dn nhn vi mt fluorochrome nh Cy3
hoc Cy5 pht hin. i vi iu tra ca b gen, ADN Geno-mic c dn nhn
trc khi n ADN. DNA
mnh v vi trnh t b sung s lin kt vi
c nh sn phm PCR v c hnh qua b la-bel. Th h ca microarray PCR da
trn lao ng
v chi ph cc khon thm canh v pht ca tp cht trong
cc sn phm PCR c th dn n lai cho v
kt qu khng r rang.
5.2.2. microarray oligonucleotide da trn
Nhng mng cung cp li th ng k so vi microarray PCR. S dng
oligonucleotide tng hp
loi b s cn thit cho RT-PCR khuch i v sn-UCT bc thanh lc v lai to c
th c mi lp c trc tip s dng tng s RNA ca vi khun, l
dn nhn trc khi lai to. C Advanta-ges khc bao gm gim nhim cho v
lai cho [286].
microarray oligonucleotide da trn theiap andhly
gene c s dng ng thi pht hin v betweenListeriaspecies dis-buc
ti [79]. Trong cc nghin cu khc,
microarray gen PCR da trn c xy dng v
c s dng phn loi L. monocytogenesstrains [285.287].

cc mi quan h pht sinh loi ofL. monocytogenessero-loi 1 / 2a, 1 / 2b, 1 / 2c v

4b c kim tra s dng
cc phng php [285.288.289] v h thng di ng cho
lnh vc th nghim cng c m t [290.291]. Cc
hu ht tnh nng hp dn ca cng ngh mi ny l
kh nng nhn dng ng thi v nh my ca
Listeriastrains trong mt th nghim, mt tnh nng mnh m m khng
cc xt nghim khc cung cp. Tuy nhin, nhng kh khn l
rng mt lng ln cc DNA mc tiu hoc RNA c yu cu
thc hin cc bi kim tra v th nghim cng sut cao l chi ph cm.
6. Kt lun
K t khi truyn qua thc phm ofListeriawas u tin
thnh lp nm 1981 c mt s bng n trong
U. Gasanov et al. / FEMS Microbiology Nhn xt 29 (2005) 851-875 865
pht trin cc bi kim tra targetingListeria mi, n reser-voirs v cc ngun
nhim. Cc u tin chnh
th nghim l xc nh cc h cha Listeria
nhim trong mi trng ch bin thc phm v
pht hin sm cc loi thc phm b nhim ngn chn
dch truyn qua thc phm v tun th cc quy nh
yu cu. C tip tc p lc c thm ra-pid v cc phng php nhy cm, l
kt qu ca cc
yu cu ca cc t chc chnh ph bo v
ngi tiu dng, trong khi ngnh cng nghip thc phm l di p lc
pht hnh cc loi thc phm trn th trng m khng h. t
c mt? khng khoan nhng? cho ofListeriain hin din

s loi thc phm cho ngi tiu dng nhiu nc c t l-c gng, iu quan
trng l phi c xt nghim nhanh vi nhy cao. Nhng xt nghim ny cng
nn tun theo cao
lng mu v c chi ph-hiu qu. nhng tiu ch
c p ng tt nht cho n nay ca cc xt nghim ELISA da trn v d nh
cc '' day''Listeriatests tip theo cho thc phm v mi trng
mu (ListeriaUnique, Tecra International, Frenchs
Rng, c v VIDASListeriaexpress. sinh hc Mrieux, Marcy Etoile, Php), hoc PCR
thng thng
kim tra v xt nghim da trn ADN (Bng 1
v 8).
c nhng ci thin ng k trong nn vn ha
phng php, c trong s la chn v kh nng phc hi
vt nhn mnh. Nhng phng php ny c s dng lm giu
mu th nghim v thng c phi hp vi
Xt nghim khng th da trn hoc thm cc xt nghim gn y phn t
onListeria c th da gen. vn ha v kim tra chng da trn c th nh vy c
s dng rng ri trong ngnh cng nghip thc phm labo-ratories v s tin li, tc
ca h,
t ng ha, lng mu cao v chi ph hiu qu-Ness (Bng 8). tin cy, mnh m
v repro-ducibility, c chng minh trong nhiu xc nhn
hc. Trong khi cc phng php ny l rt quan trng i vi vic kim hng ngy
thc phm v mi trng cc mu, cc
khng th xc nh tiu loi, m l mt quan trng
tham s trong cc nghin cu v ra-chia listeriosis.
p dng phng php vn ha v huyt thanh
nghin cu dch t hc l gi tr gii hn v
kh nng phn bit thp ca h. Phng php in dis-hnh php hc cao hn, bao
gm c phng php phn t nh vy

nh PCR v cc xt nghim ADN, devel-oped phn bit gia cc chng v xc

nh cc tiu loi ofL. monocytogenesserotypes (Bng 8). vic u tin
phng php nh my c da trn du kiu hnh nh vy
nh cc loi thc khun v tnh di ng in di ca cytosolic
enzym. Gn y, c mt xu hng
s dng phng php phn t m cn c ngha
tng cng kh nng phn bit, l mnh m, ng tin cy
v cho kt qu ti sn xut. Tiu chun ca phng php l mt vn quan trng
cho cc ng dng thng thng v c th l
t c bng cch t ng ha cc th nghim nh t ng
ribotyping, PCR v cng real-time PCR. Vi cao
chnh xc v tc m xt nghim phn t c th c
thc hin, khng c nghi ng rng hu ht cc meth-ods, nu cha c p dng,
s c thng qua bi quy nh
chnh quyn v trong cc phng th nghim kim nghim thc phm trong tng lai.
C pht trin mi th v trong phn t cng ngh cao nhng cng nh cc cng
ngh microarray c
tim nng ln lm c s kim tra thng xuyn trong tng lai.
Li cm n
U. Gasanov v P. M. Hansbro c h tr cho
cng vic ny bi TECRA Quc t, i hc
Newcastle, v Vin nghin cu Y khoa Hunter.