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PCR Sequencing PDF
PCR Sequencing PDF
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.
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
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.
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
C
G
C
A
G
T
C
C
T
A
G
C
T
T
A
G
C
G
G 5
84
3.
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
CATACGTGG CCTTACG
GGAATGC
thm
dNTP, DNA
polymerase,
ddATP, ddTTP,
ddGTP, ddCTP
CGTAAGGCCdA
CGTAAGGCdC
CGTAAGGCCACdG
CGTAAGGCCACGTdA
CGTAAGGdC
CGTAAGGCCACGTA TdG
CGTAAGGCCAdC
CGTAAGGCCACGdT
CGTAAGGCCACGTAdT
*CGTAAGGCCACGTA TdG
*CGTAAGGCCACGTAdT
*CGTAAGGCCACGTdA
*CGTAAGGCCACGdT
*CGTAAGGCCACdG
*CGTAAGGCCAdC
*CGTAAGGCCdA
*CGTAAGGCdC
*CGTAAGGdC
Chiu in di
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 [+]
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.
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.
DNA t mhim sc th
c ph bung thnh
nhiu mnh nh
Cc mnh DNA nh
c gii trnh t
c ag
cca gc a
g g cg g c
g ga g c a g
a gc
ga
g t g ag
g a c ag c
t t t cc
c g a g c g g cg g
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
acgttct
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
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.
Clone vo cc
vector
a vo cc
h thng t
bo biu hin
Lp th vin
protein
Th nghim trn cc
loi t bo ch
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
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.
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.
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
Biosubtyl DL
Bacillus subtilis
10 /gr
Bacillus subtilis
10 /gr
Bacillus subtilis
10 /gr
Bacillus cereus
10 /gr
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
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.
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
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.
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.
Rhinocladiella at rovirens
3 (5%)
X ylaria curt a
1 (2 %)
Neosart orya f ischeri
2 (3%)
A spergillus niger
4 (7%)
A spergillus f umigat us
23 (4 0%)
Schizophyllum cominune
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
Dch v nh danh vi nm
Trn c s ca k thut PCR v gii trnh t gene 28s rDNA, phng th nghim
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.
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
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
CEQ8000
(8 capillaries)
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
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
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
109
PCR khuch i an c
hiu 550bps trn vng
gene preS ca HBV
cc
Lamivudine,
bin
Adefovir
khng
v
ABI 3130XL
(16 capillaries)
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
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
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
54.28
60
50
40
30
20
10
29.89
8.79
6.59
0.45
204
204+207
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
Yu cu tm t bin
precore/promoter ca HBV
Bnh nhn c
HBeAg- HBeAb+,
HBVDNA+,
ALT - bt thng
PCR khuch i an c
hiu 250bps trn vng
gene core ca HBV
KT QU
(24 gi sau khi nhn mu)
A1762T
2
1%
9 G1896A
6% A1762T
G1896A
36
26%
Khng t bin
27
19%
G1896A
A1762T, G1764A
28
20%
38
28%
A1762T, G1764A
v nhy cm nn c th ni cng c
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%)
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 .
Kt qu pht hin
MTB trong vng 24gi
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
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
113
114