PCR v gii trnh t

Gii trnh t l g?
DNA l c s ha hc ca gen. Phn t DNA l mt chui xon kp ca hai mch n
c cu to t 4 loi nucleotide khc nhau nh cc base ca chng, l: A (adenine),
C (cytosine), G (guanine) v T (thymine). Cc nucleotide ny ni kt lin tip vi nhau
theo mt th t xc nh. Gii trnh t ca gen tc l pht hin c th t sp xp ca 4
loi nucleotide ny trn phn t DNA.
Cc phng php gii trnh t gene
1.

Phng php ha hc gii trnh t DNA

Vo nm 1977, Maxam v Gilbert pht minh c mt phng php ha hc

c th gii trnh t mt on DNA. Nguyn tc ca phng php ny l: (1) Trc

ht l phi nh du mt u ca on DNA cn phi gii trnh t bng mt gc
phospho ng v phng x (32P); (2) X l cc on DNA nh du ny vi cht
ha hc c th lm bin i c hiu mt hoc hai loi base ca nucleotide trn on
DNA. V d, dimethylsulphate bin i Guanine bng cc thm mt gc methyl v
tr nitrogen th 7 (N7), hydrazine lm bin i Thymine v Cytosine, hydrazine v
NaCl lm bin i Cytosine, acid lm bin i Guanine v Adenine, NaOH
lm bin i Adenine v Cytosine vi Adenine u tin hn Cytosine. Nh vy trong
giai on ny, phi dng t nht 4 ng nghim v trong mi ng nghim, DNA c x
l vi mt lng rt gii hn ca mt trong cc cht ha hc nu trn mi ng
nghim ch c cha cc on DNA m trn mi on DNA ny ch c mt v tr
nucleotide c hiu vi cht ha hc l b bin i; (3) Cc nucleotide b bin i ny
s b ly ra khi mch khung ng-phosphate (sugar-phosphate backbone) ca on
DNA v nh tch ra c cc on mch n c mt u nh du 32P v mt u
b mt phn t base v phn t ny b ly ra khi mch khung (hnh 37). (4) Thc
hin in di mu DNA x l trong 4 ng nghim ny trn 4 hng ca mt gel
polyacrylamide bin tnh cc mch n trong mu di chuyn trong gel khng b bin
i trong qu trnh in di. Nh , sau khi hon tt in di, cc mch n s b dng
ti cc v tr khc nhau, ty thuc vo mch n ny di ngn khc nhau, theo hng dc
sut chiu di ca gel. p gel in di ny trn mt phim nhy tia X, cc v tr dng
81

li ca cc mch n trn gel in di s to thnh cc vch trn phim v cc v tr ny

u b nh du phng x do cc mch n u c mt u c nh du bng 32P.
T cc vch trn phim x k t ghi ny (autoradiography), c th c c trnh t ca
cc nucleotide ca on DNA (hnh 38).
Phng php ha hc xc nh trnh t DNA ca Maxam v Gilbert trn thc t
khng phi d thc hin v cn phi xc nh kh nhiu thng s ti u cho th nghim,
trong kh nht l phi xc nh c nng gii hn nht ca cc cht ha hc sao
cho khi x l mu DNA th trn mi on DNA ch c mt base b bin i ng vi
mi v tr ca base b bin i th s c mt mch n c u nh du 32P c tch ra.
Chnh v s phc tp ny, phng php Maxam Gilbert hin nay t c s dng, m
thay vo cc nh khoa hc dng phng php enzyme vi nhiu u im vt tri
hn.
Mch n DNA

32

P A p G p C p T p T p T p G p A p G p G p A p C p G p A..

32

Cc mch DNA
n c u b
nh du 32P

P
P
32
P
32
P
32
P
32

Hnh 37: Cc mch n c mt u nh du vi 32P v mt u base Guanine b ly khi mch khung do b

bin i. Cc mch n ny c chiu di ngn khc nhau ty thuc vo v tr ca Guanidine trn
on DNA.

3
5
GCCCACTTCAGAAGAAAAAGG

C
T+G
G
A>C

Hnh 38: Hnh x k t ghi trn phim nhy tia X mt gel polyacrylamide sau khi in di. Cc vch l v tr cc
mch n b dng li sau in di. Cc v tr ny gn hay xa khi im l tu kch thc ca mch n
di hay ngn khc nhau. T cc vch ny, c trnh t ca on DNA trong mu.

82

2.

Phng php enzyme gii trnh t DNA

Phng php enzyme c Sanger v cc cng s pht minh cng vo nm 1977,

v ngy hm nay phng php ny cng ngy cng c hon thin v thc hin d
dng ti cc phng th nghim. Nguyn tc chung ca phng php ny c th tm tt
nh sau:

CATACGTGG

CCTTACG

mi GGAATGC

CATACGTGG

thm
d*NTP, DNA
polymerase,
ddATP

thm
d*NTP, DNA
polymerase,
ddCTP

CGTAAGG*C*CdA
CGTAAGG*C*C*A*C*G*TdA

CCTTACG
GGAATGC

thm
d*NTP, DNA
polymerase,
ddGTP

thm
d*NTP, DNA
polymerase,
ddTTP

CGTAAGG*C*C*A*CdG
CGTAAGG*C*C*A*C*G*T*A *TdG

CGTAAGGdC
CGTAAGG*CdC
CGTAAGG*C*C*AdC

CGTAAGG*C*C*A*C*GdT
CGTAAGG*C*C*A*C*G*T*AdT

in di trn gel
CGTAAGG*C*C*A*C*G*T*A *TdG
CGTAAGG*C*C*A*C*G*T*AdT
CGTAAGG*C*C*A*C*G*TdA
CGTAAGG*C*C*A*C*GdT
CGTAAGG*C*C*A*CdG
CGTAAGG*C*C*AdC
CGTAAGG*C*CdA
CGTAAGG*CdC
CGTAAGGdC

Hnh 39:

Chiu in di

Phng php Enzyme gii trnh t DNA. on DNA c chn vo mt vector ti mt v tr bit r
trnh t, nh c th tng hp c cc mch n c mt u l mi, mt u l cc nucleotide
35
tn. Trong hnh cc nucleotide nh du * l cc nucleotide c dnh du S, nh vy cc mch
n c nh du.

83

Trc ht chn cc on DNA cn phi xc nh trnh t vo mt vector l phage

hay plasmid ti mt v tr m trnh t chui ca v tr ny c xc nh r. Chuyn
th tc l a cc vector mang on chn DNA ny vo t bo vi khun nhn bn
cc vector ny thnh nhiu bn sao, sau tch chit v tinh khit cc vector t vi
khun thnh cc vector t do.
Dng cc on mi b sung mt cch c hiu vi trnh t ca vector ti v tr
chn on DNA. Thm vo ng phn ng men DNA polymerase v 4 loi nucleotide
t do (gi l dNTP: deoxynucleotide triphosphate) v mt lng rt gii hn cc
nucleotide tn (terminator, l ddNTP: dideoxynucleotide triphosphate, cng nh dNTP,
c 4 loi ddNTP tng ng vi 4 base A, T, C v G). Phn ng c thc hin trong 4
ng, v mi ng cho mt loi ddNTP khc nhau. Bt u t on mi, men DNA
polymerase s ko cc dNTP vo tng hp mch n b sung vi mch DNA chn
trn vector, v s tng hp mch n s b dng
li ti v tr m ddNTP c ko vo thay v
dNTP. L do s tng hp b dng li l v
ddNTP c cu trc ha hc b mt i gc OH ti
v tr carbon th 3 ca ng deoxyribose, m
gc OH ti v tr ny chnh l ni dNTP k
tip c gn vo. Nh vy trong ng phn ng

C
G
C
A
G
T
C
C
T
A

c cc mch n DNA c cc chiu di khc

G
C

nhau tng ng vi cc v tr trnh t cc

T
T
A

nucleotide trn on DNA gc (hnh 39).

G
C

gii trnh t, ngi ta dng phng php

in di trn gel polyacrylamide bin tnh. Vi
phng php nh du mi hay cc dNTP bng

G
G 5

Hnh 40: Hnh x k t ghi mt kt qu gii

trnh
t
DNA
trn
gel
polyacrylamide. T kt qu ny,
c c trnh t DNA

ng v phng x 32P hay 35S th sau khi in di,

cng ging nh phng php ca Maxam v Gilbert, ngi ta pht hin cc vch in
di bng k thut x k t ghi, v t cc vch ny gii c trnh t ca on DNA
(hnh 40).

84

3.

Gii trnh t bng my t ng (automated sequencer)

Cc phng th nghim hin nay u dng phn ng gii trnh t bng phng php

enzyme, nhng khi lm gii trnh t th thng dng cc my t ng ch khng dng

k thut x k t ghi nh trc y. thc hin c gii trnh t bng my t ng
th cc mch DNA n sn sinh trong ng phn ng gii trnh t phi c nh du
hunh quang cc vch in di ca cc mch n ny pht sng khi i qua mt chm
tia sng laser. Cu to ca mt my t ng gii trnh t gm hai phn chnh yu,
l: phn in di vi gel polyacrylamide v phn pht hin cc vch in di. Phn in

Mt cm quang

Tia
LASER

A G C T T A C G G A C T A A TGC
Mu 1
Mu 2

My tnh

Mu 3
Mu 4

Thpolyacrylamide.
i gian
Hnh 41: S khi mt my t ng gii trnh t dng bn gel
My gm hai phn: phn
in di gel polyacrylamide, phn pht hin cc vch in di. Cc vch in di c cc con mt cm
quang pht hin khi i qua chm tia laser v pht sng ln. Tn hiu c mt cm quang truyn v
my tnh hin th thnh cc nh cng sng
85

di polyacrylamide c th l mt bn gel hay l mt ng mao qun cha gel. Phn pht

hin vch in di l nhng con mt cm quang v mt chm tia laser i qua trc n.
Nguyn tc hot ng ca my l trong sut qu trnh in di, mi khi c mt vch
in di i qua chm tia laser th vch in di s pht sng ln v s pht sng ny s
c con mt cm quang

CATACGTGG CCTTACG
GGAATGC

ghi nhn v lu li thnh

mt nh cng sng

thm
dNTP, DNA
polymerase,
ddATP, ddTTP,
ddGTP, ddCTP

trong biu . T biu

ca cc nh cng

CGTAAGGCCdA
CGTAAGGCdC
CGTAAGGCCACdG
CGTAAGGCCACGTdA
CGTAAGGdC
CGTAAGGCCACGTA TdG
CGTAAGGCCAdC
CGTAAGGCCACGdT
CGTAAGGCCACGTAdT

sng ny, my s so dng

ca cc nh tng ng
vi cc mu cui cng
phn tch thnh trnh t
ca on DNA (hnh 41).
Vi cc my th h
mi sau ny, ngi ta c
th dng 4 mu hunh
quang khc nhau nh
du 4 loi ddNTP, nh vy
phn ng gii trnh t c
th thc hin c ch

*CGTAAGGCCACGTA TdG
*CGTAAGGCCACGTAdT
*CGTAAGGCCACGTdA
*CGTAAGGCCACGdT
*CGTAAGGCCACdG
*CGTAAGGCCAdC
*CGTAAGGCCdA
*CGTAAGGCdC
*CGTAAGGdC

Chiu in di

Hnh 42: Vi cc ddNTP nh du hynh quang khc nhau, cc an

trnh t tn cng u 5 s c nh du bng 4 mu
hynh quang khc nhau tng ng vi nuleotide tn l A,
hay T, hay C hay G. Chnh nh vy khng cn phi thc
hin phn ng gii trnh t trong 4 ng phn ng.

trong mt ng nghim v
khi gii trnh t ch cn in di trn mt hng m khng cn phi trn 4 hng khc nhau
nh trc y (hnh 42). Nu dng din di mao qun th ty thuc vo s mao qun c
th chy mt ln m ngi lm th nghim c th c s mu cho mi ln chy nhiu
hay t. Hnh 43 l s khi minh ha cho mt gii trnh t 8 mao qun (CEQ8000,
hay CEQ8800). Ty thuc vo qui m ca phng th nghim cng vi s mu gii
trnh t m phng th nghim c th trang b gii trnh t t hay nhiu mao qun. V d
vi ABI th c nhiu loi: ch 1 mao qun, 4 mao qun, 16 mao qun, 48 mao qun, ri
96 mao qun. Phng th nghim R&D ca cng ty Nam Khoa trc y nhu cu gii
trnh t khng nhiu nn ch trang b CEQ8000 c 8 mao qun (hnh 44). Nhng nay
86

do nhu cu nghin cu v dch v ngy cng nhiu nn trang b thm ABI 3130XL
c n 16 mao qun (hnh 44).
Cc th h my v sau ngy cng c nhng chng trnh i km chng nhng
c th hin th t ng cc k t ho hc ca trnh t DNA m cn gip cc nh nghin
cu c th: (1) Pht hin cc chi tit cn thit trn trnh t nh m khi u, m kt
thc, vng c m (ORF: Open Reading Frame), trnh t c hiu cho cc enzyme ct
gii hn (restriction enzyme), cc du n di truyn...m cc chi tit ny rt cn thit
lp bn gen...(2) Phin dch trnh t trong vng c m (tc l vng trnh t c
ngha) thnh trnh t acid amin ca mt protein, nh c th xc nh, phng on,
hay lm c s ban u hiu c chc nng ca gen...(3) T ng so snh trnh t
DNA trong th nghim hay
mt trnh t DNA np vo
my, hay c th trnh t acid

Cm
quang

amin ca protein, vi cc

Cc [+]

trnh t khc c trong ngn

hng d liu pht hin

Mao
qun

c s tng ng vi trnh
t bit, nh lm c s
xc nh gen v chc nng
gen...

Laser

Cc [-]

in th cao

Hnh 43: S khi mt my din di mao qun dng gii trnh t DNA

Hnh 44: My gii trnh t CEQ8000 c 8 mao qun ca Beckman Coulter v ABI 3130XL c 16 mao qun
ca Applied Biosystem c trang b ti phng th nghim R&D ca cng ty Nam Khoa

87

PCR l cng c gip hon thin v m rng ph p dng k thut gii trnh t
PCR ng gp rt nhiu vo cng ngh gii trnh t lm cho cng ngh gii trnh t
ngy cng d dng hn v thun tin hn. Khng ch vy PCR cn lm cho gii trnh t
c nhiu ng dng hn khng ch trong nghin cu m c trong chn on na.
Trc khi c PCR th ngi ta ch c th gii trnh t cc on gene c c lp v
chn vo plasmid v ch c nh vy th mi c th nhn bn c on gene mun gii
trnh t thnh nhiu bn sao vi s lng chy c phn ng gii trnh t. Ngoi
ra, enzyme polymerase cng l loi enzyme khng chu nhit nn phn ng gii trnh t
ch thc hin 37oC, do vy m vn ti u cc thnh phn ca phn ng rt l kh
ng thi hiu qu ca phn ng gii trnh t cng khng cao.
T khi c s ra i ca k thut PCR th k thut gii trnh t cng c nhng tin b
rt vt bc. Ch cn dng PCR l c th nhn bn on gene mun gii trnh t thnh
hng t bn sao hon ton ging ht nhau v sn phm khuch i ny c th a vo
gii trnh t trc tip m khng cn phi chn vo mt plasmid gii trnh t na. S pht
minh ra k thut nhn bn DNA trong ng nghim bng PCR cng gip cho vic ci tin
phn ng gii trnh t km hiu qu trc y tr nn hiu qu hn nh p dng enzyme
polymerase gii trnh t bn vi nhit phn ng gii trnh t c thc hin qua cc
chu k nhit ging ht PCR, m ngy nay chng ta gi l phn ng chu k nhit gii trnh
t. Vi phn ng chu k nhit gii trnh t th hiu qu phn ng tt hn rt nhiu, ng
thi ti u ha cc thnh phn ca phn ng cng d dng hn rt nhiu. C th ni
chnh s ra i v hon thin k thut PCR gip tng tc k thut gii trnh t khng
nhng trong vn hon thin n, m c trong vn phm vi p dng.
Gii trnh t b gene ngi
1.

Lm th no gii trnh t b gene ngi

Gii trnh t b gen ngi tc l gii trnh t ton b th t sp xp cc nucleotide

A, T, C, v G ca DNA trong 23 cp nhim sc th t bo ngi. D n gii trnh t b

gen ngi c Tin S James Watson, cha ca pht hin cu trc DNA, ra t
gia thp nin 1980. n thng 10 nm 1990, d n c ra i vi kinh ph 3 t M
kim do HGP (Human Genome Project) ca Vin Quc Gia Y T Hoa K lnh o
cng vi s tham gia ca nhiu trung tm nghin cu b gen ngi ca nhiu quc gia
88

trn th gii. Do tin on gi tr kinh t mang li t vic gii m thnh cng b gen
ngi s rt ln, nn c mt s cng ty sinh hc chuyn v b gen ngi c
hnh thnh. S nhp cuc ca cc cng ty t nhn ny to nn mt bu khng kh thi
ua cho vic khai ph b mt v b gen ca loi ngi chng ta, cng gn n nhng
ngy n ch cng cc k ngon mc. Trong cuc chy ua ny, nh p dng mt
phng php cc k c o kt hp gia cng ngh PCR, cng ngh gii trnh t
DNA vi cng ngh tin hc, nn ch vi kinh ph khong 200 triu m kim, Tin S
Crag Venter ca Celera Genomics tr thnh ngi dn u thnh cng nht trong vic
gii trnh t c gn nh hon ton b gen ngi.
Ci kh khn ca vic gii trnh t b gene ngi l khng th no tch tng i
phn t DNA ca tng nhim sc th ri em gii trnh t i phn t DNA nguyn
vn ny. Cc nh khoa hc ca HGP cng nh cc trung tm khc trn th gii nghin
cu v b gen ngi thng ct cc i phn t DNA ca nhim sc th mt cch nh
hng theo bn di truyn, ri em gii trnh t cc on ct DNA ny, v cui cng
tm cch ghp ni chng li vi nhau cho ng v tr ca chng trn i phn t DNA.
Phng php ny i hi phi tn nhiu thi gian v cng sc v c nhiu khi cc on
ct DNA qu ln, rt kh gii trnh t cng nh kh cho kt qu gii trnh t chnh xc.
Do , vi mi mt on DNA cc nh khoa hc phi thc hin gii trnh t t nht l
7 ln m bo lp li. Chnh v vy, vi cch tip cn ny, nhiu v tr trn cc
i phn t DNA vn khng th gii trnh t c m t cc nh khoa hc ca HGP
c th hon tt vic xp ng v tr cc on DNA gii trnh t trn i phn t
DNA ca nhim sc th.
Tin s Crag Venter thc hin mt phng php gii trnh t b gen ngi mt
cch hon ton khc hn (hnh 45). Trc ht ng cho tch chit ton b DNA nhim
sc th t bo ngi ri ph bung mt cch khng nh hng cc i phn t DNA
thnh cc mnh DNA nh hn. em gii trnh t tt c cc mnh DNA nh ny. Cui
cng dng nhng my in ton cc mnh m ng hp tc vi cng ty ABI ch to
ra, d tm cc u trng lp ca cc mnh DNA nh ny lp ni vo ng cc v tr
ca chng trn i phn t DNA ca nhim sc th. Chnh nh p dng chin lc gii
trnh t ny m Celera Genomics gii trnh t rt nhanh cc mnh DNA, ch cn lp
li cho mi mnh DNA c 5 ln, v xp ng gn nh hu ht v tr ca cc mnh
89

DNA gii trnh t trn i phn t DNA ca nhim sc th. Hn th na, TS. Crag
Venter cng tuyn b cng ty trin khai mt k thut mi c th lm cho phng
php gii trnh t b gen ca cng ty, vn d l phng php nhanh nht ri, tr
thnh nhanh hn na. Cui nm 2000, Celera Genomics hon thnh vic gii trnh
t b gen ca chut vi 2.3 t k t ha hc trong c rt nhiu tng ng vi b
gen ngi, nh vy s gip cc nh khoa hc d dng hn trong vic truy tm cc chc
nng ca cc gen.
2.

Kt qu gii trnh t b gene ngi

Sau gn 10 nm nghin cu

thc hin vi nhng nm cng v

sau cng si ng khng kh thi

DNA t mhim sc th
c ph bung thnh
nhiu mnh nh

ua do s nhp cuc ca cc cng

ty t nhn, ngy 26 thng 6 nm
2000, ti Ta Bch c vi s

Cc mnh DNA nh
c gii trnh t

hin din ca Tng Thng Hoa

c ag

K Bill Clinton, hai khoa hc gia

cca gc a

din cho Celera Genomic v Tin

Institute of Health) cng b mt

g g cg g c

g ga g c a g

a gc

ga
g t g ag

g a c ag c

Cc mnh DNA nh c chng trnh phn tch

t ng tm cc u trng lp, nh ti hin
c trnh t ca i phn t DNA

S Francis Collins i din cho

Project) ca NIH (NIH: National

t t t cc

c g a g c g g cg g

M l Tin S Crag Venter i

HGP (HGP: Human Genome

g ca

g a ga c a g c
g g ag
c c a gc a t
g
g
a
c
g
g
a
g g c gg c
cc a g c
c a g g ca t t t
tgtac
c g a g c g g cg g
a g a c a gc
g t g ag g
cc a g c a
t
t
a
t
g
g c
gg c g g a g c a
c ga gc g g c

ccgtcaaccggagtta

cch long trng l chng trnh

gii trnh t b gen ngi

acgttct

hon tt. y chnh l mt thi

khc lch s cc k quan trng v
ng nh ca nhn loi, nh du
ngy nhn loi c trong tay
mt bn sinh hc cn thit
bc vo k nguyn mi vi
nhiu ha hn cc thnh tu v

Hnh 45:
Phng php gii trnh t b gen ngi ca cng ty Celera
Genomics: trc ht tch chit ton b DNA ca nhim sc
th t bo ngi, sau ph tung DNA thnh cc mnh nh,
gii trnh t tt c cc mnh nh ny, cui cng nh chng
trnh vi tnh cc mnh ni kt cc on DAN nh ny li
vi nhau bng cch d tm cc u trng lp nh vy ti hin
c mch DNA nguyn thy

90

i v sinh y hc trong tng lai. D thnh cng ca cc nh khoa hc gii trnh t

c 3.1 t cc k t ha hc lm nn b gen ngi c v nh s kin lch s
con ngi bc ln mt trng (ngy 26 thng 7 nm 1969, t khoang b phi thuyn
Apollo, Neil Armstrong bc xung i do trn mt trng v nh du thi im con
ngi thc hin c hon ton c m tng ao c by lu: khc du chn mnh
trn b mt ch Hng), nhng y cha phi l thi im m con ngi toi nguyn
c c m hiu bit tng tn c b gen ca mnh.
Tht s cho n thi im cc nh khoa hc Cng ty Celera Genomic hy
cn cha gii trnh t c 3% b gen ngi, tc khong 150 triu, v cng vic ny
i hi phi t 1 n 2 nm na mi c th hon tt v 3% cn li ny nm trn phn
kh gii trnh t nht. Cn HGP th thnh cng t hn, hy cn nhiu on trnh t ca
b gen m cc nh khoa hc ca HGP cha gii c v cng c nhiu on trnh t
gii c nhng h vn cha sp xp vo ng v tr. Ngoi ra, cho d cc nh khoa
hc c gii trnh t c mt cch trn vn b gen con ngi, th cun sch ca i
sng (the book of life) ny hy cn cha c c, nh Gerald Rubin, Ph Gim c
Nghin Cu Y Sinh Hc ca Vin Y Hc Howard Hughes gii thch: Cun sch c
vit bng mt ngoi ng rt phc tp, cn phi c mt thi gian di chng ta mi hiu
c. Tht vy, c th tng tng l nu cc trnh t ca b gen ngi c in ra
thnh sch th s cn phi khong 200 cun v mi cun dy phi 500 trang. C 3000
trang nm ri rc trong 200 cun sch trn l c ngha tc l c gen, m cho n hin
nay ch mi c mt s t trang trong 3 ngn trang ny l khoa hc c c tc l
xc nh c gen. Hy cn vi trang (khong 3%) cha vit xong v nhng trang ny
l nhng trang kh vit nht v thuc nhng vng kh gii trnh t nht trong b gen
ngi.
Cho n thng 4 nm 2003, cc trang khuyt ny c cc nh khoa hc vit
hon tt. Tuy vy, mt vic rt ln hin ang t ra trc mt cc nh khoa hc l
phi xc nh c cc trnh t no l gen v chc nng ca cc gen ny. Ch c 3%
ca b gen ngi l mang gen tc l mang trnh t c ngha v hin nay cc nh khoa
hc cng cha bit chnh xc l tht s c bao nhiu gen trong b gen ngi. Cu tr
li phng chng nht l t 28.000 n 140.000 gen, v gn y nht c l l ang gn
n con s chnh xc hn, l 30.000 gen. Trong khong 30.000 gen ny, cc nh
91

khoa hc hin ch bit c chc nng ca khong 10.000 gen, hy cn 20.000 cha
bit chc nng ca chng. y chnh l mt cuc ua mi cng ha hn y si ng
v kt qu tng chng mt ca cuc ua, tc kt qu ca vic xc nh c chc nng
ca tng gen, u ha hn nhng p dng cc k ngon mc v y trin vng trong
sinh hc cng nh y hc.
3.

Gii trnh t b gene ngi cuc ua cha kt thc

Gen chnh l trnh t cc nucleotide trong chui DNA ch huy c s tng hp

mt protein. Chnh v vy xc nh c chc nng ca mt gen tc l xc nh c

protein m gen tng hp c cu trc nh th no v c nhim v g trong c th sng.
Sau kt qu gii trnh t b gen ngi, hin nay cc nh khoa hc ang c nhiu chin
lc c th nhanh chng pht
hin chc nng ca cc gen trong
b gen ngi. Chng ti xin trnh
by ra y ba trong s nhng chin
lc .

Dng PCR tng

hp cc gen

Clone vo cc
vector

(1) Nh kt qu gii trnh t

b gen ngi, cc nh khoa hc s
nhanh chng xc nh cc trnh t

a vo cc
h thng t
bo biu hin

mang gen. Vi cc k thut sinh

hc phn t hin i nh k thut

Lp th vin
protein

PCR, cc nh khoa hc s d dng

nhn bn cc gen ny trong ng

Th nghim trn cc
loi t bo ch

nghim ri dng ha chng vo cc

vect sau nh cc h t bo
biu hin biu hin cc protein.
Vi cch ny cc nh khoa hc s
nhanh chng thnh lp c mt

Hnh 46: Chin lc to th vin protein tm chc nng

ca gen, tm tt l t gen tng hp cc protein, tr
trong th vin protein, ri th trn cc h thng t bo
ch pht hin chc nng ca protein, t bit
c chng nng ca gen.

th vin cc protein ca cc gen c trong b gen ngi. Vn tip theo l phi xc

nh cc protein trong th vin protein l c chc nng g? bit c chc nng ca
protein trong th vin, cc nh khoa hc s th nghim cc protein ny h thng cc
loi nui cy t bo tm hiu mi giao tip gia protein th nghim vi t bo ch,
92

nh xc nh t bo ch no c phn ng vi protein th nghim, v nh vy s

bit c chc nng protein bit c chc nng ca gen. (hnh 46).
(2) Gii trnh t b gen ca ng vt hu nh
nh d nhn (rt ging b gen ngi) hay chut,
y l nhng b gen ca cc ng vt c nhiu
tng ng vi b gen ngi, nhng li trn cc
ng vt m cc nh khoa hc rt d lm th
nghim. Trn b gen ng vt ny, cc nh khoa
hc s d dng pht hin cc gen, v cc chc nng
ca gen thng qua s thay i hoc biu hin ca
mt tnh trng no ca ng vt th nghim mt
khi cc nh nghin cu dng cc k thut sinh hc

Hnh 47: Hnh dng ca phn t khng th

c vi tnh ha

phn t gy t bin hay lm knock-out ngay trn nhng vng mang gen ca b gen.
Mt khi xc nh c gen v chc nng ca mt gen trn ng vt th nghim, cc
nh nghin cu s d dng truy tm v pht hin gen v chc nng ca gen ny trn b
gen ngi nh pht hin s tng ng v trnh t trong gen ngi vi gen ca ng
vt th nghim.
(3) Chng ta bit rng mt protein c tng hp t gen hot ng nh mt vai tr
cu trc hoc vai tr chc nng l nh hnh dng ca n vi cc hc nh hay cc np
gp. Chnh v vy cho nn d mt gen c c gii m v biu hin c mt protein,
th cng cha th xc nh c chc nng ca protein ny nu cha bit c cc
hnh dng ca n. Chnh v vy m Vin Quc Gia Khoa Hc Y Khoa Tng Qut ti
Hoa K ang chi mt khong 20 triu la thnh lp cc trung tm Proteomics, l
cc trung tm chuyn nghin cu v lu tr cc cu trc v hnh dng c vi tnh ha
(hnh 47) ca cc protein c trong thin nhin. Cc vin Proteomics nh vy cng
ang v s thnh lp ti cc nc khc. Cc nh khoa hc hy vng rng trong vng 10
nm ti y, cc vin proteomics s lu tr c hnh dng ca gn 10.000 protein c
trong thin nhin. Con s ny d hy cn rt nh so vi mt s lng kh ln cc
protein thc s c trong thin nhin, nhng cc nh khoa hc cng hy vng rng th
vin ny sc bao gm tt c hnh dng ca tt c cc protein c lin quan n sinh
hc v y hc, v cc protein khc khng c trong danh sch cng ch l cc bin th hay
93

cc ng dng vi cc protein c trong danh sch m thi. Nh cc trung tm

proteomics v th vin cc hnh dng protein, cc nh nghin cu chc chn s d dng
hn trong xc nh cc chc nng ca gen.
4.

Vit Nam c th tham gia vo cuc ua ny khng?

Hin nay cc nh khoa hc ang c gng nghin cu khai ph cc b mt trong

cun sch i sng ghi chp y trnh t b gen ngi vi nhiu trin vng s c
cc ng dng cch mng trong sinh hc, y hc, cng nh cc lnh vc khoa hc khc
v s a n nhiu tin b mi cho loi ngi trong k nguyn mi ny. Cc nh ysinh hc ca chng ta hon ton c th tham gia trong cuc ua ny, v theo chng ti
th sinh hc phn t i vi cc quc gia thu nhp thp c th coi nh l mt ngnh
khoa hc tiu th cng nghip hin i. Tiu th cng nghip v khng nht thit
phi c cc my mc cc k hin i v tn km m vn c th thc hin c cc k
thut bng cc thit b khng qu t tin. Hin i v nh nghin cu thc hin k
thut da trn cc kin thc hin i vi cc kt qu hon ton c th c ng gp ln
vo kho tng kin thc hin i ca nhn loi. Vn chnh l chng ta phi c chun
b nh th no v phi c chin lc tham gia vo cuc ua nh th no.
chun b, quan trng nht l chng ta phi c gng xy dng v o to trong
cng nh ngoi nc i ng cc nh nghin cu y sinh hc c trnh v bn lnh
ngang tm vi th gii. Cc nh nghin cu ca chng ta khng ch phi c kin thc
v k nng v chuyn mn sinh-y hc m cn phi c kh nng tip cn khai thc
c kho tng kin thc cng nh cc d liu ngy cng cp nht trn mng internet.
Ngoi ra, chng ta phi trnh tnh trng o to cc nh nghin cu nhng li khng
chun b cho h cc iu kin cng nh phng tin lm vic. Chnh v s thiu thn
iu kin cng nh phng tin lm vic m cc nh nghin cu ca chng ta d c
trnh v bn lnh n u cng s thui cht dn nhit huyt cng nh kh nng theo
thi gian, v dn dn tnh trng chy mu cht xm trong cng nh ngoi nc s
khng th trnh khi.
V chin lc, th c l chng ta cng c gng tham gia cuc ua vi nhng ch
nhm l gii quyt v khai thc nhng c th trong nc. Chn hn khai thc nghin
cu c ch mc sinh hc phn t tc ng ca cc dc liu trong nc bng cch
xy dng cc m hnh th nghim ex-vivo trn cc cy t bo pht hin s cm ng
94

biu hin gen khi cho cc h thng nui cy t bo ny tip cn vi cc trch phn ca
dc liu. Pht hin cc c ch tc ng ca dc liu mc sinh hc phn t, chc
chn chng ta s khai thc c cc ngun dc liu qu m chng ta vn c sn trong
nc. Ngoi ra, dn tc Vit ca chng ta l mt dn tc kh thun nht, bn cnh
chng ta cng c nhng dn tc t ngi sng rt tp trung v cha h b lai trn, y
chnh l c s cho cc nghin cu v s phn b cc gen cng nh cc m di truyn
cho cc nghin cu ng dng sau ny pht hin, cha tr hoc phng nga cc bnh
l di truyn.
Nhn loi ang bc vo thin nin k 21 vi hnh trang mang theo l cc tin b
vt bc nhiu lnh vc khoa hc khc nhau, trong c khoa hc Y hc v Sinh
hc. Mt hnh trang cn yu nht cho mt cuc sng d dng hn v tt p hn l
cun sch ca i sng vi hu nh ton b trnh t ca b gen ngi m cc nh khoa
hc mi va kp hon tt. Vic khai thc cun cm nang sinh hc cn yu nht ny ca
nhn loi i hi mt s hp tc a ngnh cng nh chuyn ngnh. Chnh v vy nn
chng ti cho rng chng ta cng khng th b qua yu t hp tc v hc hi: chng ta
phi hp tc v hc hi t bn b quc t, v quan trng nht l phi bit hc hi v
bit hp tc gia chng ta vi nhau.
ng dng cng ngh gii trnh t ti phng th nghim NK-Biotek
Phng th nghim NK-Biotek v n v R&D ca cng ty Nam Khoa ng dng kh
thnh cng k thut gii trnh t vo cc dch v chn on v nghin cu. Di y xin
a ra mt s thnh tu tiu biu.
1.

ng dng k thut PCR v gii trnh t nh danh vi khun

Nh pht hin c mt cp mi m sau c t tn l

NK

16s-F v

NK

16s-R

khuch i c on DNA di 527 bps cha cc trnh t gip phn bit ging v loi
cc vi khun, phng th nghim NK-Biotek v n v R&D ca cng ty Nam Khoa
xy dng thnh cng qui trnh PCR cho gene 16s rDNA v qui trnh gii trnh t trc
tip sn phm PCR ny nh danh vi khun. Thnh cng ny gip phng th
nghim NK-Biotek v n v R&D ca Nam Khoa thc hin c kh nhiu ng dng
trong cc xt nghim dch v v nghin cu.

95

a.

ng dng trong xt nghim vi sinh lm sng nh danh vi khun k kh

Vi khun k kh l cc vi khun ch c th tng trng trong iu kin kh trng

khng c oxygene phn t (O2). Vi khun k kh c th l cc vi khun c li c s

dng trong cng ngh vi sinh, tuy nhin v mt y hc rt c nhiu loi vi khun k kh
l cc tc nhn nhim khun ni sinh hay ngoi sinh. Bnh l nhim khun k kh rt a
dng, t cc nhim khun ti ch kt hp vi cc vi khun khng k kh, hay cc nhim
khun h thng, hay cc nhim khun c hiu. Do a dng nh vy nn vic xc nh
mt nhim khun k kh v mt lm sng khng phi d dng. Trong khi xt nghim
vi sinh lm sng ti cc phng th nghim thng b hn xt nghim vi khun k kh v
gp phi nhiu tr ngi v k thut nh l: (1) C c phng tin chuyn ch bnh
phm sao cho gi c bnh phm lun iu kin k kh cho n khi n c phng
th nghim. (2) To kh trng k kh nui cy phn lp c vi khun k kh t cc
bnh phm. (3) nh danh cho c vi khun k kh. (4) Lm c khng sinh vi
khun k kh.
Trong cc tr ngi trn, tr ngi kh gii quyt nht l vn nh danh vi khun
k kh phn lp c t cc bnh phm. C nhiu b th nghim nh API 20A ca BioMerieux, RapIDTM ANA II ca Oxoid, PRAS II (Scott), AN Ident (Analytab), ANA
card (Vitek)...c sn trn th trng cho cc phng th nghim chuyn dng cho nh
danh vi khun k kh. Tuy nhin cc b th nghim ny kh c sn ti Vit Nam v nhu
cu khng nhiu. Hn na cc b th nghim ny cng c nhiu hn ch nh t l nh
danh sai kh cao (19% i vi API 20A, 10% i vi RapIDTM ANA II), t l khng
th nh danh n loi cng nhiu (38% i vi RapIDTM ANA II, 29% i vi API
20A).
Chnh v vy nn c th thc hin c xt nghim vi sinh lm sng vi khun
k kh, phng th nghim NK-Biotek v n v R&D ca cng ty Nam Khoa trin
khai mt gii php ton din, bao gm ch to c mi trng chuyn ch CaryBac
c th gi bnh phm iu kin k kh trong thi gian 24 gi hn c thi gian cn
thit chuyn mu t lm sng n phng th nghim, s dng n nh phng tin
to kh trng k kh bng cc ti Anaerocult A ca Merck, thc hin c khng sinh
vi khun k kh bng b th nghim khng sinh k kh do n v R&D nghin
cu v thit k ra. Trong nh danh vi khun k kh, phng th nghim NK-Biotek v
96

n v R&D ca cng ty Nam Khoa a ra mt tip cn hon ton mi v hin i,

nhng li t tn km nht, l gii php gii trnh t mt on cha trnh t c hiu
ging v loi nm trong gene 16S ca vi khun.
C th tm tt qui trnh nh sau: Vi khun mc trn hp thch phn lp c gt
vo nc mui sinh l v khun t c 0.5 1 McF. Sau un cch thy si
trong 10 pht. ngui ri ly tm 13.000RPM trong 5. Ly 5ml dch ni cho vo PCR
mix 45ml cha sn cc thnh phn k c cp mi NK16s-F v NK16s-R khuch i on
DNA di 527 bps cha trnh t c hiu loi trn gene 16S ca vi khun. Chy chng
trnh nhit PCR gm 1 chu k 95oC/5 kch hot enzyme hot start Taq polymerase,
ri 40 chu k 94oC/15-60oC/30-72oC/1. Sn phm PCR sau c tinh sch bng
b Wizard SV Gel and PCR clean-up Sytem ca Promega. Sn phm tinh sch c
in di trn thch v sau nh lng bng quang ph GenQuant ca Pharmacia, sau
c a vo phn ng gii trnh t 2 chiu s dng mi xui v mi ngc ca
PCR thc hin trn b thuc th DTCS cycle sequencing kit ca Beckman Coulter.
Chng trnh lun nhit ca phn ng gii trnh t vi b thuc th trn l 30 chu k
96oC/20-50oC/20-60oC/4. Sn phm ca phn ng gii trnh t c ta bng
ethanol, ri sau c chy in di gii trnh t trn my gii trnh t CEQ8000. Kt
qu gii trnh t c so chui bng chng trnh blast search trn ngn hng d liu
gene ca NCBI. T kt qu so chui, chng ta s c c kt qu nh danh vi khun.
Sau ny, vi cc trang b thm nh in di bng DNA chip ca Bio-analyzer v my
gii trnh t ABI 3130XL 16 capillary, qui trnh ca phn ng gii trnh t v in di
gii trnh t cng c nghin cu tng thch vi cc trang b ny. C th l
thay v in di trn gel ri nh lng bng quang ph 260/280 th kt hp c hai bng
in di v nh lng trn Bio-analyzer DNA chip ca Agilent; thay v dng DTCS
l phn ng gii trnh t th dng BigDye Terminator V. 3.1 Cycle Sequencing kit
ca ABI vi chng trnh lun nhit ca phn ng gii trnh t l trc ht 1 chu k
96oC/1, theo sau 25 chu k 96oC/10-50oC/5-60oC/4. Sn phm ca phn ng gii
trnh t sau khi ta bng ethanol, c chy in di gii trnh t trn my gii trnh t
ABI 3130XL vi gel POP 7 v mao qun 50cm. Hnh 48 l mt kt qu nh danh
bng gii trnh t 16s rDNA vi khun k kh phn lp t mu m xoang ca bnh nhn.
Phng th nghim NK biotek p dng tip cn ny trong nm 2008 cho xt nghim
97

vi sinh lm sng vi khun k kh vi cc mu bnh phm l 39 mu m xoang ly t

bnh nhn b vim xoang mn tnh. Kt qu xt nghim cho thy c 31% (18/59) cy ra
tc nhn vi khun k kh v kt qu nh danh bng phng php gii trnh t 16s
rDNA cho thy c 11% (2/18) Veillonella (cu khun Gram -), 17% (3/18) Prevotella
buccae (trc khun Gram -), 22% (4/18) l Propionibacterium acnes, 6% (1/18) l
Streptococcus aginosus, 17% (3/18) l Streptococcus constellatus, 17% (3/18) l
Streptococcus gordonii, 6% (1/18) l Peptostreptococcus micros, v 6% (1/18) l
Peptostreptococcus stomatis. Kt qu ny cho thy PCR v gii trnh t 16s rDNA l
mt gii php k thut hon ton c th s dng nh danh vi khun k kh, gii
quyt c khu k thut phc tp v kh khn nht trong qui trnh xt nghim vi sinh
lm sng vi khun k kh.

Hnh 48: Kt qu gii trnh t 16s rDNA trc khun Gram [+] phn lp k kh t bnh phm m xoang ly t bnh
nhn cho php kt lun y l Propionibacterium acnes

b.

Pht hin cht lng cc sn phm probiotic cho ngi v cho thy sn
Probiotic chnh l cc vi sinh vt khng gy bnh, c dng lm sn phm tr

sinh qua ng n hay ung s dng trn ngi, gia sc, hay thy sn pht huy hiu
qu chng li cc nhim trng nh cc c ch nh cnh tranh cht dinh dng v ch
98

bm ca vi sinh vt gy bnh, phc hi vi khun ch bnh thng cho rut hay cho mi
trng, kch thch h thng min dch khng c hiu ca c th vt ch chng li vi
sinh vt gy bnh, hay tit ra cc cht c tnh khng sinh khng c cc vi sinh vt
gy bnh.
Cc vi khun thng c dng lm probiotic l Bacillus subtilis, Lactobacillus
acidophilus, Bifidobacterium...v thng c cung cp di dng bt ghi hm lng
v cc loi vi khun c trong sn phm. Do cc vi khun ny thng l cc vi khun d
mc nn ch vi cc phng tin phng th nghim v cc mi trng nui cy thng
thng l c th sn xut c cc sn phm probiotic nn hin nay trn th trng ca
chng ta xut hin kh nhiu sn phm probiotic c sn xut t rt nhiu c s trong
v ngoi nc s dng khng ch cho gia sc, thy sn m c cho ngi. Chnh v
tnh trng ny nn c mt vn m nhiu ngi rt quan tm, l liu cht lng
ca cc sn phm probiotic hin ang c mt trong th trng c tht s ng nh nh
sn xut cng b hay khng, c bit l liu cc vi khun c ghi trong nhn hiu sn
phm c ng l vi khun c trong sn phm hay khng, v hm lng c m bo
ng nh vy hay khng.
tr li cu hi trn, trong nm 2008 phng th nghim NK-Biotek v b phn
R&D ca Nam Khoa hng dn mt s ti tt nghip lun vn Thc S c ni
dung tm hiu cht lng cc sn phm probiotic dng cho ngi v thy sn hin
ang lu thng trn th trng. kim tra hm lng th gii php k thut khng
kh, ch cn thc hin cy nh lng bng phng php thch hay phng php
lng trn b mt mi trng dinh dng v iu kin thch hp l c th cho c
kt qu nh lng chnh xc hm lng vi khun ch c trong mu sn phm cn
phi kim tra. Tuy nhin xc nh chnh xc tn vi khun c trong sn phm c
ng nh tn c nu trong nhn sn phm hay khng li l mt vn khng n
gin v cc vi khun probiotic nh B. subtilis, L. acidophilus...khng phi l cc vi
khun d dng c nh danh n loi nu ch da vo cc tnh cht sinh vt ha hc.
Chnh v vy, chng ti gii php PCR v gii trnh t gene 16s rDNA ca vi khun
phn lp c t cc sn phm probiotic xc nh danh tnh ca vi khun. Kt qu
cho thy c kh nhiu loi sn phm probiotic s dng cho ngi v cho thy sn c
vn v mt cht lng c v hm lng ln v vi khun tht s c trong sn phm.
99

Bng 13 v 14 trnh by kt qu cho thy s khc bit gia vi khun tht s c trong
sn phm probiotic so vi kt qu c nh sn xut cng b trn nhn.
Bng 13: Kt qu nh danh bng k thut PCR v gii trnh t 16s rDNA v kt qu nh lng cc vi khun
tht s c trong cc probiotic s dng trn ngi
STT

Tn sn phm

Tn vi khun trn nhn sn phm

Kt qu PCR v gii trnh t 16sDNA

6

Biosubtyl DL

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

Bidisubtilis (gi giy)

Bacillus subtilis

10 /gr

Bacillus cereus

10 /gr

Bidisubtilis (gi nhm)

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

Bio Acimin

Bacillus subtilis

10 /gr

Bacillus cereus

10 /gr

Biosubtyl II (gi)

Bacillus subtilis

10 /gr

Bacillus pumilus

10 /gr

Biosubtyl II (vin)

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

Subtyl

Bacillus subtilis

10 /gr

Bacillus cereus

10 /gr

Medilac Vita

Bacillus subtilis

10 /gr

Bacillus cereus

10 /gr

Bio Baby

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

10

Bio Vita

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

7
6
7
7
8
7
6
6
6

Bng 14: Kt qu nh danh bng k thut PCR v gii trnh t 16s rDNA v kt qu nh lng* cc vi khun
tht s c trong cc probiotic s dng cho thy sn
STT

Tn sn phm

Tn v lng* vi khun
trn nhn sn phm

DT-LACTO

Bacillus subtilis
Lactobacillus acidophilus

Bio-DW

Bacillus subtilis
Lactobacillus acidophilus

Probiotex-one

Bacillus subtilis

Kt qu nh danh
v nh lng* thc t
Bacillus pumilus
Khng tm thyL. acidophilus
9

10 /gr

Bacillus subtilis
Khng tm thyL. acidophilus

10 /gr

B. megaterium
11

Bacillus subtilis

10 /gr

Vime-Subtyl

Bacillus subtilis

10 /gr

Biozyme for fish

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

Novazyme F

Bacillus subtilis

10 /gr

Bacillus subtilis

10 /gr

Subtyl-LA

Bacillus subtilis

NPV-Prozyme 90

Bacillus subtilis
Lactobacillus acidophilus

10 /gr
9

ProKura

Bacillus subtilis

10

Men gu vng

Bacillus subtilis
Lactobacillus acidophilus

11

Biobac

Bacillus subtilis

10 /gr

Bacillus subtilis
Khng tm thyL. acidophilus

10 /gr

Bacillus subtilis

10 /gr

6
7

Bacillus cereus
Khng tm thyL. acidophilus
Bacillus pumilus
11

Bacillus subtilis
Khng tm thyL. acidophilus

10 /gr

Bacillus subtilis
Khng tm thyL. acidophilus

10 /gr

12

Pond clear

Bacillus subtilis
Lactobacillus acidophilus

10 /gr

13

Bio Pond

Bacillus subtilis
Lactobacillus acidophilus

10 /gr

14

Men Bac

Bacillus subtilis
Lactobacillus acidophilus

15

Microzyme

Bacillus subtilis
Lactobacillus acidophilus

16

Zymmix

Lactobacillus acidophilus

17

Lactovet

Lactobacillus acidophilus

Zeofarm

Bacillus pumilus

18

Lactobacillus acidophilus

Bacillus cereus
Khng tm thyL. acidophilus
?

Bacillus subtilis
Khng tm thyL. acidophilus

10 /gr

Acidovorax sp.
Clostridium phylofermentant
9

10 /gr

L. acidophilus

10 /gr

*Ch nu ln lng cc vi khun m nh danh bng PCR v gii trnh t cho thy kt qu khng sai lch

100

Phn tch cc d kin trnh by trong hai bng trn, chng ta thy i vi cc sn
phm probiotic dng cho ngi, s sai bit v hm lng gia kt qu kim tra so vi
cng b trn nhn khng nhiu (khng qu 10 ln), nhng c n 50% tn vi khun
cng b khng ng vi kt qu nh danh. Thm ch c n 4 sn phm li s dng B.
cereus, vi khun khng c php c mt trong thc phm v c kh nng gy tiu
chy v i ma do c th cha cc toxin, lm vi khun tr sinh. i vi cc sn phm
probiotic dng cho thy sn, kt qu kim tra cho thy i vi vi khun B. subtilis, c
6/15 mu khng phi l B. subtilis m l B. cereus hay B. pumilus; v ch c mt sn
phm l c cha L. acidophilus cn hu ht l khng tm thy vi khun ny nh cng
b trn nhn. Hnh 49 minh ha mt kt qu gii trnh t gene 16s rDNA xc nh vi

Hnh 49: Mt chng vi khun phn lp t mt sn phm probiotic nu chi da vo tnh cht khun lc v nhum
Gram th khng th bit y khng phi l B. subtilis, nhng khi gii trnh t 16s rDNA th xc nh c
y l B. cereus.

khun l B. cereus trong khi nhn sn phm li l B. subtilis. Cn v mt hm lng th

tht thm hi, trong cc cc sn phm c kim tra tht s c vi khun nh cng b
th a s hm lng tt gim t 104 n 105 so vi cng b. Kt qu ca nghin cu l
mt ting chung bo ng cc nh sn xut cng nh cc c quan kim tra cht
101

lng nn quan tm n vn cht lng ca cc sn phm probiotic s dng cho

ngi v thy sn hin ang lu hnh ti Vit Nam; v kim tra c cht lng
cc sn phm probiotic th phi s dng PCR v gii trnh t gene 16s rDNA v ch c
gii php ny mi c th nh danh chnh xc vi khun c s dng lm probiotic,
trnh nhm ln vi cc vi khun khng c tc dng probiotic hay thm ch c th c
hi.

Hnh 50: Mt phiu l lch 16s rDNA ca mt chng vi khun dng trong kim chun vi cc thng tin v tnh cht
khun lc, hnh nh Gram, cc tnh cht sinh ha c bn, v quan trng nht l trnh t gene 16s rDNA

102

c.

Xy dng l lch 16s rDNA ca cc chng vi khun dng trong kim chun
Trong ni kim hay ngoi kim vi sinh, rt cn thit phi dng cc chng vi sinh

c l lch nh danh chng chnh xc. Cc phng th nghim vi sinh thng yu cu

cung cp cc chng quc t vi l lch xut pht t nh cung cp chng. Hin nay rt
kh c th t mua cc chng gc quc t v vn nguy c khng b sinh hc.
Chnh v vy gii php hay nht hin nay l s dng cc chng gc quc t hin ang
lu gi ti cc phng th nghim, thm ch s dng cc chng gc phn lp c
nhng phi c l lch dnh danh chnh xc n loi. ng trc yu cu ny, phng th
nghim NK-Biotek v n v R&D ca Nam Khoa xy dng l lch 16s rDNA ca
tt c cc chng quc t ATCC, cng nh mt s chng phn lp c v tiu biu cho
cc ging v loi vi khun cn cho kim chun. Hnh 50 minh ha mt l lch 16s
rDNA ca 1 chng vi khun c phng th nghim NK-Biotek v n vi R&D ca
Nam Khoa cung cp dng trong kim chun.

Hnh 51: Mt kt qu nh danh bng PCR v gii trnh t 16s rDNA ca mt chng vi sinh cng nghip cho
thy y l Acrobacterium tumefaciens, l vi khun rt kh nh danh bng vi sinh kinh in

103

d.

Lm xt nghim dch v nh danh vi khun

Cng vi cng ngh PCR v gii trnh t 16s rDNA ca vi khun, phng th

nghim NK-Biotek v n v R&D ca Nam Khoa v ang thc hin dch v nh

danh cc vi khun c dng trong vi sinh cng nghim cng nh vi sinh nng nghim
c gi n t cc cng ty, nh my, hay t cc yu cu ca cc ti nghin cu.
Mu c gi n nh danh c th l cc chng vi khun cy trn mi trng nui
cy, hay l cc dch tch chit DNA, hay huyn dch vi khun. C th ni vi gii php
k thut PCR v gii trnh t gene 16s rDNA, cc vi khun d l n u, d kh nh
danh n u cng u c nh danh n loi. Hnh 51 l kt qu mt chng vi
khun dng trong vi sinh cng nghim c yu cu nh danh, v kt qu nh danh
cho thy y l vi khun Agrobacterium tumefaciens.
2.

ng dng k thut PCR v gii trnh t nh danh vi nm

Trong cc xt nghim vi sinh th nh danh vi nm c l l mt tr ngi ln nht v

nu s dng cc phng php kinh in th khng ch phn tch hnh thi i th v vi

th, vn d phc tp v cng phu, m cn phi lm cho c cc kho st sinh vt ha
hc khc nh ng ha ng, phn tch cc cht bin dng...Chnh v vy thng
thng t c phng th nghim vi sinh lm c xt nghim vi nm.
Nhn din c tr ngi k thut chnh yu ny, phng th nghim NK-Biotek v
n v R&D cng ty Nam Khoa c gng tm gii php vt qua, v sau mt thi
gian nghin cu - th nghim, xy dng c mt gii php k thut l s dng
PCR v gii trnh t gene 28s rDNA. Nguyn tc ca gii php ny l s dng b
NK

FUNGI-DNAPREP do n v R&D ca cng ty Nam Khoa ch to tch chit

DNA ton phn t mu vi nm hay mu th cha vi nm cn nh danh, sau s

dng PCR vi mt cp mi c t tn l

NK

28s-F v

NK

28s-R c hiu mt on

DNA di khong 260 bps c cha cc trnh t c hiu ging v loi ca vi nm trn
gene 26s rDNA, v sau cng l gii trnh t trc tip v 2 chiu sn phm PCR nh
danh vi nm qua blast search trn ngn hng d liu gene ca NCBI.
ng dng gii php k thut ny, phng th nghim NK-Biotek v n v R&D
ca cng ty Nam khoa ang thc hin rt thnh cng hai dch v, l:

104

a.

Pht hin v nh danh vi nm trong cc bi nm ly ra t phu thut bnh nhn

vim xoang
Trc y, cc bnh phm bi nm (fungus ball) thng c cc bc s tai mi

hng gi n phng th nghim gii phu bnh v kt qu thng c nhn v sau 1

tun l nhn thy vi th cc si t nm nghi Aspergillus. Hin nay vi xt nghim PCR

Penicit rium cit rinum

3 (5%)
M alassezia rest rict a
2 (3%)

Rhinocladiella at rovirens
3 (5%)

X ylaria curt a
1 (2 %)
Neosart orya f ischeri
2 (3%)
A spergillus niger
4 (7%)

Cand ida ort hopsilosis

1 (2%)

A spergillus f umigat us
23 (4 0%)

Schizophyllum cominune
1 (2%)

Pichia guilliermo ndii

1 (2%)

Pseudallescheria boydi
4 (7%)

A spergillus f lavus
2 (3%)

A spergillus oryzae
11 (19%)

Biu 12: Tng kt cc kt qu pht hin v nh danh nm bng k thut PCR v gii trnh t 28s rDNA trong
cc mu bi nm ly t m vim xoang trn bnh nhn

v gii trnh t c thc hin ti phng th nghim NK-biotek th cc nh lm sng s

c kt qu sau 2 ngy vi nh danh vi nm n loi. Biu 12 tng kt cc kt qu
pht hin v nh danh vi nm trong cc bi nm c bnh vin Tai Mi Hng TP.H
Ch Minh gi n trong thi gian gn y. C tng cng 58 mu c gi n lm xt
nghim, v c 58 u pht hin v nh danh c vi nm l tc nhn gy bnh.
b.

Dch v nh danh vi nm
Trn c s ca k thut PCR v gii trnh t gene 28s rDNA, phng th nghim

NK-Biotek v n v R&D ca cng ty Nam Khoa trin khai c xt nghim dch v

nh danh vi nm vi cc ngun mu l t cc cng ty, nh my trong lnh vc cng
nng nghip, hay l t cc phng th nghim ca cc trng i hc hay ca cc trung
tm nghin cu. C th ni l cho n nay, hu nh khng c mu vi nm no gi n
phng th nghim NK-Biotek l khng c kt qu nh danh. Hnh 52 v hnh 53 minh
ha hai kt qu nh danh nm trong xt nghim dch v nh danh vi nm ca NKBiotek.
105

Hnh 52: Vi nm c hnh dng vi th hnh ht men nh gc trn tri cho kt qu PCR v gii trnh t 28s
rDNA nh bn di, v t trnh t ny kt qu blast search trn ngn hng gene NCBI cho kt
qu nh danh Candida tropicalis vi tng ng 99%.

Hnh 53: Vi nm c hnh dng vi th hnh que di nh gc trn tri cho kt qu PCR v gii trnh t 28s
rDNA nh bn di, v t trnh t ny kt qu blast search trn ngn hng gene NCBI cho kt
qu nh danh Rhinocladiella atrovirens vi tng ng 99%.

3.

ng dng PCR v gii trnh t pht hin, nh lng v xc nh genotype

HCV
Pht hin, nh lng v xc nh genotype HCV l cc thng s m bc s cn

phi bit trc khi cho ch nh iu tr c hiu bng interferon phi hp vi ribavirin
l pht iu tr chun cho bnh nhn c antiHCV [+]. Pht hin HCV-RNA xc
nh l bnh nhn ang nhim HCV, nh lng HCV-RNA l xc nh lng virus
trc khi bt u iu tr v lm c s theo di hiu qu iu tr, cn xc nh
genotype HCV l gip bc s iu tr quyt nh c thi gian iu tr cho bnh
nhn. Thng thng cc phng th nghim lm sng s phi thc hin cc yu cu ny
ring l, ngha l h phi pht hin HCV-RNA trc, ri mi nh lng, v cui cng
106

mi nh genotype HCV v thng th

do yu cu k thut nn cc xt

Yu cu xc nh
genotype HCV

nghim ny u phi bt u t mu
huyt thanh ca bnh nhn. Cng ty
Nam Khoa li s dng mt tip cn
khc rt mi m, tin li, v t tn km

nh lng HCV-RNA
bng xt nghim
RT-qPCR s dng kit
NK
RT-HCVqPCR

hn pht trin mt gii php ton

din thc hin c cc yu cu ny
ca bc s cng mt lc. l gii
php s dng RT real-time PCR

Xc nh genotype HCV: gii trnh t trc tip sn

phm HCVqPCR (#200bp trn vng 5NC)

pht hin v nh lng HCV-RNA

trong mu huyt thanh bnh nhn v
sau gii trnh t trc tip sn phm
real-time PCR xc nh genotype
HCV bng cch so chui trnh t gii

CEQ8000
(8 capillaries)

c vi ngn hng d liu genotype

HCV trn th vin NCBI v th vin

KT QU
24 gi sau khi nhn mu

Los Alamos.
xc nh genotype HCV, ngoi

ABI 3130XL
(16 capillaries)

nh lng

37.017 copies/ml

Kiu gene

1c

l do l mt mt xch ca gii php

ton din nh nu trn, cng ty
Nam Khoa chn tip cn gii trnh t
v cn c nhiu l do: (1) Nu s dng
kit InoLIPA ca Siemen (Bayer) l kit
c cc nh y hc tha nhn th s l
mt la chn kh tn km v gi thnh
ca kit ny kh cao, kt qu c i khi
khng phn bit c subtype, v vi

Hnh 54: Qui trnh RT-realtime PCR v gii trnh t vng

5NC pht hin, nh lng v nh genotype
HCV

version c th c s nhm ln gia 6a v 1b; (2) Nu s dng k thut gii trnh t

vng 5NC ca HCV bng h thng gii trnh t kn Trugene ca Siemen (Bayer) d
rt c nhiu nh y hc tha nhn, nhng cng s l mt la chn tn km v gi
107

thnh ca cc vt liu tiu hao i km theo h thng ny rt cao; (3) S dng phng
php real-time PCR vi cc probe c hiu l mt tip cn n gin v r tin nhng
khng nhy cm cho chn on v vng khuch i khng phi l vng 5 NC (l
vng c cng nhn l nhy cm nht cho chn on), khng c kh nng phn bit
cao cc subtype thng gp v kh thit k c cc probe c hiu n subtype, v
nguy c pht hin cho khng c hiu m vn c lm tng l c kh nng pht
hin ng nhim.
Chnh v cc l do trn, Phng th Nghim Y Khoa NK-BIOTEK v cng ty Nam
Khoa nghin cu v xy dng c phng php xt nghim xc nh genotype
HCV bng k thut gii trnh t vng 5NC ca HCV nhng thay v s dng h thng
gii trnh t kn ca Trugene, s dng h thng gii trnh t m ca ABI (my ABI
3130XL c 16 mao qun) hay ca Beckman Coulter (my CEQ8000 c 8 mao qun).
Tip cn ny mang n cho bnh nhn v cc nh lm sng nhiu li im: (1) Nh
gii trnh t trc tip c sn phm PCR t real-time PCR s dng mi v taqman
probe c hiu vng 5NC nn kt qu xt nghim xc nh genotype lun i km vi
kt qu nh lng HCV-RNA. Do vy cc nh lm sng trc khi quyt nh iu tr
c hiu cho bnh nhn ch cn cho mt ch nh xt nghim xc nh genotype HCV
l m khng cn cho thm xt nghim nh lng. (2) t c nhy cho chn
on nh sn phm ch gii
trnh t xc nh genotype HCV
chnh l sn phm PCR da trn
vng ch 5NC l vng duy nht
trn genome HCV c tha
nhn l nhy cm lm chn
on xc nh v nh lng
HCV. (3) t c chnh xc
trong xc nh genotype HCV
khng khc bit vi h thng gii
trnh t kn Trugene nh gii

Biu 13: Phn b cc genotype HCV trong 2000 mu

huyt thanh bnh nhn nhim HCV c gi
n xt nghim ti phng th nghim NK-Biotek
trong nm 2008

trnh t ng ngay on ch m h thng Trugene s dng trn vng 5-NC ca

genome HCV. (4) Nh lm sng hay nghin cu cn c cung cp thng tin v trnh
108

t vng 5NC gii c v cc thng tin ny rt qu gi trong cc nghin cu v

phylogenetic gp phn trong nghin cu ngun gc nhim trng hay nghin cu
ng nhim HIV/HCV.
Hnh 54 tm tt qui trnh pht hin, nh lng v nh genotype HCV bng k
thut RT real-time PCR trn ch l vng 5 NC ca b gene HCV ri sau gii trnh
t trc tip sn phm PCR xc nh genotype HCV. Trong nm 2008, tng kt 2000
mu xt nghim nh genotype HCV thc hin ti phng th nghim NK-Biotek, chng
ti nhn thy a s genotype ca HCV nhim trn bnh nhn l 1b (58%), k l 6a
(17%). Biu 13 tng kt s phn b cc genotype HCV trong 2000 mu huyt thanh
bnh nhn nhim HCV c gi n xt nghim ti phng th nghim NK-Biotek
trong nm 2008.
4.

p dng PCR v gii trnh t pht hin t bin khng thuc ca HBV
Hin nay ti Vit Nam, nh cc tin b v k thut sinh hc phn t nh lng

c HBV lm c s nh gi hiu qu iu tr; v pht cng nh cc thuc c

kh nng iu tr bnh vim gan virus B mn tnh lun c sn trn th trng bc s
d dng la chn, nn vic iu tr c hiu cho bnh nhn vim gan virus B mn tnh
ph bin hn. Tuy nhin, iu tr c hiu bnh l vim gan virus B mn tnh bng
cc thuc khng virus l mt iu tr lu di. Chnh v vy nn HBV (virus vim gan B,
hepatitis B virus) c c hi tip xc lu di vi cc thuc khng virus v to c cc
t bin khng thuc, v t y ny sinh ra yu cu phi c xt nghim pht hin cc
t bin khng thuc ca HBV, c bit i vi cc thuc nh lamivudine, adefovir v
entecavir l cc thuc thng c s dng hin nay.
pht hin y cc t bin khng cc thuc nu trn, nu chn tip cn realtime PCR hay PCR-RFLP th ch c th pht hin c rt t t bin khng thuc ca
HBV v c qu nhiu cc bin th ca HBV lm cho vic thit k cc probe pht
hin t bin hay thit k cc v tr enzyme ct gii hn nhn din b kh khn rt
nhiu. Hn na cho n hin nay, y vn ch mi bo co c real-time PCR v PCRRFLP pht hin t bin khng lamivudine hai v tr 180 v 204 trong khi cn
nhiu t bin na khng ch to cho virus khng lamivudine, m c adefovir v
entecavir na. Chn tip cn nh vy chc chn s khng p ng c thc t.

109

Do vy phng th nghim NKYu cu tm t bin

khng thuc ca HBV

BIOTEK v cng ty Nam Khoa

chn mt tip cn khc da trn
phng tin v trang thit b hin

PCR khuch i an c
hiu 550bps trn vng
gene preS ca HBV

i ang c cng ty, chnh l

tip cn PCR v gii trnh t mt
on DNA di 550 bps trn vng
gene preS lm xt nghim pht
hin

cc

Lamivudine,

bin
Adefovir

Tm t bin khng thuc: gii trnh t trc tip sn

phm PCR 550bps t vng gene preS

khng
v

Entecavir. Xt nghim da trn tip

ABI 3130XL
(16 capillaries)

cn ny hin nay ang c nhiu

KET QUA (24 gi sau khi nhan mau)

nh lm sng v nghin cu y hc
tin cy nh cc u im sau: (1)
Pht hin c hu ht cc v tr
t bin c th xy ra gy ra
khng 3 loi thuc trn; (2) Vi
nhy cm ca thit b gii trnh t

TAATTTGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATC
TTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATC
AACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGG
AAATTGCACTTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTACTA
GTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAAC
ATCTTGAATCCCTTTTTACCGCTGT

hin nay (sequencer ABI 3130XL,

gii hai chiu), khng ch tt c cc
im t bin k trn u c gii
n, m c tnh trng heterozygote
ca t bin/hoang di vn c
pht hin; (3) Vi trnh t gii
c, kt qu xt nghim cn cung
cp thng tin mt cch chnh xc
v genotype ca HBV m khng
cn phi c ch nh xt nghim

Genotype
Pht hin t bin
Khng
Lamivudine
I169T
V173L
L180M
A181T
T184S
M204I
V207M/I
Q215S

KQ
K
K
Co
K
K
Co
K
K

Genotype C
Khng
Adefovir
L80V/I
S85A
V84M
A181V/T
V214A
Q215S
N236T

KQ
K
K
K
K
K
K
K

Khng
Entecavir
T184G
S202I
M250V

KQ
K
K
K

Hnh 55: Qui trnh xt nghim pht hin t bin khng

thuc v xc nh genotype ca HBV bng k
thut PCR v gii trnh t mt an DNA 550 bps
trn gene PreS ca HBV

ny t lm sng. Hnh 55 m t qui trnh xt nghim PCR v gii trnh t gene preS
pht hin cc t bin khng thuc v xc nh genotype HBV, trong kt qu pht
hin t bin c trnh by trong mt bng lit k cc v tr t bin t bin khng

110

lamivudine, adefovir v entecavir c cc nh nghin cu ghi nhn v tt c cc v

tr t bin ny u nm trong trnh t c
gii.

54.28

60

Trong nm 2008, phng th nghim

50

NK-Biotek ca cng ty Nam Khoa thc

40

hin c 455 xt nghim PCR v gii trnh

30

t vng gene PreS ca HBV, kt qu cho

20

thy c n 69% cc trng hp l thuc

10

genotype B v 31% l genotype C, khng

c cc genotype khc. C 46% pht hin c

t bin khng lamivudine vi 30% v tr

29.89

8.79

6.59
0.45

204

204+207

204+180 204+180+207 KhongB

Biu 14:
S phn b t l cc v tr t bin khng
lamivudine pht hin c

204, 9% hai v tr 204 v 207, 6.5% 204 v 180, v 0.5% t bin c 204, 180 v
207. Kt qu ny c trnh by trong biu 14.
5.

p dng PCR v gii trnh t pht hin t bin precore/ promoter v genotype
HBV
Trong qu trnh phn ng vi cc p ng min dch t bnh nhn, HBV c th b

t bin precore v t bin core promoter. t bin precore l t bin ti v tr

nucleotide 1896 t G thnh A (G1896A) lm cho codon TGG tr thnh TAG l mt
stop codon. t bin ny lm cho HBV khng th tng hp c HBeAg. Do vy,
khi th mu bnh nhn, khng pht hin c HBeAg m HBV-DNA vn tn li cng
vi HBeAb [+]. y l mt t bin rt quan trng cn phi c pht hin v bnh
nhn s c nguy c cao vo x gan hay ung th gan, c bit khi pht hin c thm
t bin core promoter ti hai v tr nucleotide 1762 v 1764 (A1762T, G1764A). Khi
bnh nhn b pht hin mang HBV c cc t bin ny th phi c iu tr bng
thuc khng virus sut i. Chnh v tm quan trng i vi lm sng nh vy nn
trong thi gian qua phng th nghim NK-BIOTEK & cng ty Nam Khoa xy dng
c k thut PCR v gii trnh t vng gene core lm xt nghim pht hin t
bin precore v t bin core promoter v khng ch vy, cn cho bit thm v
genotype HBV. Hnh 56 minh ha qui trnh ny.
Xt nghim ny hin nay ang c nhiu nh lm sng v nghin cu y hc quan
tm v tin cy. Trong nm 2008, phng th nghim NK-Biotek v cng ty Nam Khoa
111

 thc hin 134 mu xt nghim t

Yu cu tm t bin
precore/promoter ca HBV

Bnh nhn c
HBeAg- HBeAb+,
HBVDNA+,
ALT - bt thng

bin precore v core promoter ca

HBV trn cc bnh nhn b bc s iu
tr nghi ng c t bin precore/core

PCR khuch i an c
hiu 250bps trn vng
gene core ca HBV

promoter. Kt qu cho thy c n 73%

pht hin t bin vi nhiu kiu t

Tm t bin precore/promoter: gii trnh t trc

tip sn phm PCR 250bps t vng gene core

bin khc nhau, c th ch t bin

precore, c th ch t bin core

ABI 3130XL (16 capillaries) hay

CEQ8000 (6 capillaries)

promoter, c th t bin c promoter

KT QU
(24 gi sau khi nhn mu)

v core promoter. Cc thng tin ny s

rt hu dng cc nh y hc c th
nghin cu su hn v mi lin h vi
lm sng, bnh hc v sinh bnh hc.
Biu 15 trnh by t l phn b cc
kiu t bin pht hin c.
6.

PCR v gii trnh t pht hin t

bin khng rifampicine v INH
ca M. tuberculosis
T nm 1996, chng ti bt u

Hnh 56: Qui trnh PCR v gii trnh t pht

hin t bin precore/promoter

trin khai k thut PCR pht hin M.

tuberculosis v cho n hin nay xt

A1762T

nghim PCR pht hin M. tuberculosis

2
1%

9 G1896A
6% A1762T
G1896A

36
26%

do chng ti pht trin ny c cc

Khng t bin

27
19%

nh y hc trong nc chp nhn nh

nhy cm rt cao so vi kho st trc
tip v nui cy. Vi PCR, kt qu pht
hin M. tuberculosis c th n tay nh
lm sng ch 24 gi sau khi gi mu i

G1896A
A1762T, G1764A

28
20%

xt nghim. Nh u im nhanh chng

38
28%
A1762T, G1764A

v nhy cm nn c th ni cng c

Biu 15: T l phn b cc kiu t bin

precore/core promoter pht hin
c trong 134 mu xt nghim

PCR mang n mt li ch khng

112

405
427
Leu Ser Gln Phe Met Asp Gln Asn Asn Pro Leu Ser Gly Leu Thr His Lys Arg Arg Leu Ser Ala Leu
CTG AGC CAA TTC ATG GAC CAG AAC AAC CCG CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG GCG CTG
Pro
CCG

Leu
CTA

Val
GTC

/..../
Dele

Leu
TTG

(3%)

(3%)

(9%)

(1%)

(1%)

Tyr
TAC
Asp
GAC
Gln
CAG
Asn
AAC
Arg
CGC
Pro
CCC

Leu
TTG
Gln
CAG
Trp
TGG

Pro
CCG
(1%)

(52%)

(28%)

Hnh 57: Vng nng vi cc t bin trn rpoB gene v t l thng xy ra

chi ci trong lnh vc chn on lao, vt qua cc cng c vi sinh kinh in. Mt vn
hin nay ang c cc nh y hc quan tm l lm th no c th c c kt qu
khng sinh vi khun M. tuberculosis vi thi gian ngn hn, v vi phng php vi
sinh kinh in th kt qu khng
sinh ch c th c c sau

Yu cu
PCR pht hin MTB
Tm t bin khng Rifampicine v INH

hn 2 thng vi 1 thng c
c vi khun mc c trn
mi trng nui cy v 1 thng
c kt qu khng sinh .

PCR khuch i trnh

t c hiu cha cc
t bin trn rpoB,
katG, v inhA

PCR pht hin MTB

da trn khuch i
trnh t 249bp trn
IS6110

Thng tin v khng sinh m

bc s cn bit nht l liu vi

Gii trnh t pht hin t bin

rpoB, katG v inhA

Kt qu pht hin
MTB trong vng 24gi

khun phn lp trn bnh nhn

c khng vi thuc khng

t bin rpoB
L430P
S431G
Q432K/L
433 ins TTC(phe : F)
D435G/A/V
440 ins TCGGGGTTG
H445N/D/Q/S/R/L/Y/F
S450L
L452P
I480V

lao rifampicin, c coi l hiu

qu nht cho n hin nay hay
khng c th tip tc duy tr
hay ch nh cho bnh nhn
cng

thc

khng

lao

t bin katG
S315T

t bin inhA
C-15T

rifampicine.
Trc thc t i hi ny,
phng th nghim NK-Biotek
v n v R&D cng ty Nam

Kt qu khng sinh MTB

72 gi sau khi ly mu m

Hnh 58: M t qui trnh PCR v gii trnh t pht hin cc

t bin khng rifampicin v INH ca vi khun M.
tuberculosis trc tip t mu m m khng phi qua
nui cy. Kt qu n tay cc nh lm sng ch 72 gi
sau khi ly mu m

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Khoa nghin cu v th nghim mt qui trnh PCR v gii trnh t khuch i

mt vng nng ca gene rpoB ca M. tuberculosis c th pht hin c tt c cc
t bin thng xy ra to cho vi khun khng c rifampicine (hnh 57).
Ngoi vng nng ny, chng ti cng thnh cng trong xy dng mt qui trnh
pht hin khng INH bng k thut PCR v gii trnh t gene katG pht hin v
tr t bin thng gp l S315T v gene inhA xc nh v tr t bin C-15T. Hin
nay qui trnh ny c chng ti p dng pht hin trc tip cc t bin khng
thuc ny t bnh phm m khng cn thit phi qua nui cy. Hnh 58 minh ha tm
tt qui trnh c phng th nghim NK-Biotek thc hin pht hin MTB v cho
lun kt qu khng sinh ch 72 gi sau khi ly mu m t bnh nhn. Hin nay
chng ti ang tip tc nghin cu c th c thm cc kt qu khng sinh pht
hin cc t bin khng cc thuc khng lao khc nh ethambutol, PAS, PZA,
Ofloxacin... c th p ng c mt cch y cc yu cu n t cc nh lm
sng trong cuc chin u chng li tnh hnh lao a khng ngy cng trm trng hin
nay.

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